NEUROENDOCRINOLOGY
Aromatase Inhibition Exacerbates Pain and Reactive
Gliosis in the Dorsal Horn of the Spinal Cord of
Female Rats Caused by Spinothalamic Tract Injury
Samar Ghorbanpoor, Luis Miguel Garcia-Segura, Ali Haeri-Rohani,
Fariba Khodagholi, and Masoumeh Jorjani
Department of Cell and Molecular Biology (S.G.), Department of Animal Biology (A.H.-R.), School of
Biology, College of Science (S.G.), University of Tehran, Tehran, Iran; Consejo Superior de
Investigaciones Científicas (L.M.G.-S.), Instituto Cajal, E-28002 Madrid, Spain; Neurobiology Research
Center (F.K., M.J.), Shahid Beheshti University of Medical Sciences, Tehran, Iran; and Department of
Pharmacology, Faculty of Medicine (M.J.), Shahid Beheshti University of Medical Sciences, Tehran, Iran
Central pain syndrome is characterized by severe and excruciating pain resulting from a lesion in
the central nervous system. Previous studies have shown that estradiol decreases pain and that
inhibitors of the enzyme aromatase, which synthesizes estradiol from aromatizable androgens,
increases pain sensitivity. In this study we have assessed whether aromatase expression in the dorsal
horns of the spinal cord is altered in a rat model of central pain syndrome, induced by the unilateral
electrolytic lesion of the spinothalamic tract. Protein and mRNA levels of aromatase, as well as the
protein and mRNA levels of estrogen receptors ␣ and ␤, were increased in the dorsal horn of female
rats after spinothalamic tract injury, suggesting that the injury increased estradiol synthesis and
signaling in the dorsal horn. To determine whether the increased aromatase expression in this pain
model may participate in the control of pain, mechanical allodynia thresholds were determined in
both hind paws after the intrathecal administration of letrozole, an aromatase inhibitor. Aromatase inhibition enhanced mechanical allodynia in both hind paws. Because estradiol is known to
regulate gliosis we assessed whether the spinothalamic tract injury and aromatase inhibition regulated gliosis in the dorsal horn. The proportion of microglia with a reactive phenotype and the
number of glial fibrillary acidic protein–immunoreactive astrocytes were increased by the injury in
the dorsal horn. Aromatase inhibition enhanced the effect of the injury on gliosis. Furthermore,
a significant a positive correlation of mechanical allodynia and gliosis in the dorsal horn was
detected. These findings suggest that aromatase is up-regulated in the dorsal horn in a model of
central pain syndrome and that aromatase activity in the spinal cord reduces mechanical allodynia
by controlling reactive gliosis in the dorsal horn. (Endocrinology 155: 4341– 4355, 2014)
entral pain syndrome (CPS), a neurological condition
caused by a lesion or disease of the central somatosensory nervous system (1), is associated with severe
hyperalgesia, and allodynia (2– 4). Although some interventions such as pharmacological treatment with amitriptyline and gabapentin can provide relief from central pain,
many patients do not respond to these treatments (5, 6).
The development of effective long-term treatments for
CPS is contingent on an understanding of its pathophysiological mechanisms.
C
Wang and Thompson (7) generated a rat model of CPS
by making unilateral lesions on the spino-thalamic tract
(STT). STT injury in rats results in allodynia and hyperalgesia and is associated with the activation of microglia
and astroglia, which may contribute to the secondary
damage that causes long-term dysfunction in pain perception (8, 9). Indeed, spinal glial cells are believed to be
involved in pathological pain (10). These cells have been
identified as key factors in the sensory component of
chronic pain and in the response to direct injuries of the
ISSN Print 0013-7227 ISSN Online 1945-7170
Printed in U.S.A.
Copyright © 2014 by the Endocrine Society
Received February 21, 2014. Accepted August 4, 2014.
First Published Online August 8, 2014
Abbreviations: CNS, central nervous system; CPS, central pain syndrome; ER, estrogen
receptor; Iba1, ionized calcium-binding adapter molecule 1; STT, spino-thalamic tract.
doi: 10.1210/en.2014-1158
Endocrinology, November 2014, 155(11):4341– 4355
endo.endojournals.org
4341
4342
Ghorbanpoor et al
Aromatase Control Gliosis and Pain
central nervous system (CNS) and pain development (11,
12). Previous findings suggest that the ovarian hormone
estradiol, which regulates neuroinflammation by actions
on astrocytes and microglia (13), modulates pain sensitivity (14, 15) and decreases allodynia in the STT injury
model, together with the down-regulation of glial activation (16).
In addition to being a circulating hormone, estradiol is
locally synthesized in the CNS by the enzyme aromatase
(17–20). Locally produced estradiol participates in the
regulation of synaptic function, synaptic plasticity, and
behavior (17–20). In addition, different forms of CNS injury result in an increased expression of aromatase in the
damaged region (21–23). This enhanced expression of
aromatase represents an endogenous neuroprotective
mechanism, given that the inhibition of the enzymatic activity or the silencing of the enzyme results in increased
neuronal damage (24 –29). In addition, previous studies in
quail have shown that local synthesis of estradiol in the
spinal dorsal horn plays a role in modulating pain transmission (30, 31).
Given that estradiol is locally produced in the spinal
cord of mammals (32) and estrogen receptors (ER␣ and
ER␤) are also expressed in the mammalian spinal cord
(33–36), the possibility exists for an endogenous action of
locally produced estradiol on pain processing also in mammals. Our aims in the present study were to determine
whether 1) the expression of aromatase in the rat spinal
cord is regulated by the STT injury, 2) the inhibition of
aromatase enzymatic activity after STT injury affects mechanical allodynia, 3) the inhibition of aromatase enzymatic activity after STT injury affects reactive gliosis in the
dorsal horn of the spinal cord, and 4) changes in mechanical allodynia correlate with changes in reactive gliosis in
the dorsal horn.
Materials and Methods
Animals
Experiments were performed on healthy adult female
Sprague-Dawley rats weighing 200 –250 g. Rats were grouphoused (three to four per cage) in a temperature-controlled
room, with 12:12 hours light/dark schedule and received food
and water ad libitum. Special care was taken to minimize suffering and reduce the number of animals used to the minimum
required for statistical accuracy. All surgical procedures and experiments were executed in accordance with the Guide of the
Care and Use of Laboratory animals approved by the ethical
committee of Neurobiology Research Center of Shahid Beheshti
University of Medical sciences (Tehran, Iran).
Endocrinology, November 2014, 155(11):4341– 4355
Experimental groups
This study was divided into two experiments. In the first experiment rats were divided in two groups. The control group
(sham-laminectomy) received laminectomy without electrolytic
lesion (N ⫽ 40). In the second group (STT) animals were subjected to laminectomy and STT electrolytic lesion (N ⫽ 40).
Animals were killed at days 3, 7, 14, 21, and 28 after surgery.
Samples from the eight animals per experimental group and day
were analyzed by RT-PCR and samples from six of the eight
animals were analyzed by Western blotting.
In the second experiment rats were divided into four groups.
The first group was composed of intact animals that were used
for behavioral studies (N ⫽ 6). The second group (sham-laminectomy-vehicle) received laminectomy, intrathecal cannulation, and vehicle injection, without electrolytic lesion (N ⫽ 30).
The third group of animals (STT-vehicle) was subjected to laminectomy, the electrolytic lesion, intrathecal cannulation, and
daily intrathecal injection of hydroxypropyl cellulose (0.3% in
water) as a vehicle of letrozole (N ⫽ 30). In the fourth group
(STT-letrozole), rats received daily intrathecal injection of letrozole (N ⫽ 30). Animals were killed at days 0 (intact animals), 3,
7, 14, 21, and 28 after surgery. Animals from days 0, 3, 7, 14, 21,
and 28 were subjected to open field and mechanical allodynia
tests (N ⫽ 6 per day and experimental condition). Then, after
tests, the subjects were perfused and spinal cord prepared for
immunohistochemistry (see Immunohistochemistry for ionized
calcium-binding adapter molecule 1 and glial fibrillary acidic
protein).
Estrous cycle monitoring
Estrous cycle stage was monitored by analysis of cell types in
vaginal smears. Daily vaginal smears were collected in the morning for at least eight consecutive days and allowed to dry on
microscope slides. Slides were then fixed with ethanol and
stained with toluidine blue for cell type identification. Only rats
in the estrus phase of the estrous cycle were selected for surgery.
Surgery was performed in the morning of the estrous day. The
estrous cycle was not monitored after surgery.
Spinal cord injury
Rats were anesthetized using Ketamine/Xylazine95 (60/20
mg/kg, ip). Spinothalamic tract injury (STT) was induced according to the method described by Wang and Thompson with
a small modification (12, 7). Briefly, a dorsal laminectomy was
performed at spinal segments T8-T9. After exposure of the spinal
cord, the dura was incised using a thin tip iris scissor. Then, a
tungsten electrode (5 ␮m tip, 1M⍀) was targeted to the right
STT, based on stereotaxic coordinates (laterality to midline: 0.5–
0.7 mm and depth: 1.6 –1.9 mm). Lesion was made by a brief
current pulse (300 ␮A, 90 seconds) passed through the electrode.
Electrical current was raised gradually up to 300 ␮A in 10 seconds to reduce animal reflex and encourage more consistent outcome. After surgery, all animals received 1 mL of saline (sc) to
balance electrolytes, as well as Penicillin G (im) to prevent infection. Besides, during the surgery and recovery from anesthesia, the rats were covered with warm sterilized towels. Rats were
maintained in single cages in a temperature-controlled room at
25°C. Animals showing unilateral or bilateral impairment of the
hind paw movements after sham-laminectomy or STT were excluded from the study.
doi: 10.1210/en.2014-1158
endo.endojournals.org
4343
Intrathecal cannulation surgery
Western blotting
For intrathecal drug administration, a sterilized polyethylene
catheter (PE10) was inserted in the subarachnoid space of the
spinal cord under ketamine (60 mg/kg) and xylazine (20 mg/kg)
anesthesia. The catheter was passed caudally 8 cm from the cisterna magna to the L5 vertebra (37). When 4 cm of the catheter
were inserted to up to T7 vertebra, STT injury was performed on
T8-T9 spinal cord level. Then extant tube was inserted and immediately intrathecal injection of drugs/vehicles was performed.
Western Blotting was performed on dorsal horn of T8 –10
spinal segments at the lesion site. Immediately after tissue removal (n ⫽ 6 rats for each group), dorsal half of spinal tissues
were snap frozen and then homogenized using Trizol reagent
according to the manufacturer’s protocol (Invitrogen). Proteins
were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes (Millipore). The membranes were blocked in Tris-buffered saline
containing 0.1% Tween 20 and 2% enhanced chemiluminiscence advance blocking reagent (Amersham), and then incubated overnight at 4°C with primary antibodies diluted in Trisbuffered saline, with 0.1% Tween 20 and 5% BSA. The
following primary antibodies were used: a rabbit polyclonal antibody to ER␣ (Reference MC20; Batch number F2012; Santa
Cruz Biotechnology; diluted 1:500), a rabbit polyclonal antibody to ER␤ (Reference H-150; Batch number C2213; Santa
Cruz Biotechnology; diluted 1:1000), and a rabbit polyclonal
antibody to aromatase (diluted 1:2000). The aromatase antibody was generated against a 15 amino acid–peptide corresponding to residues 488 –502 of mouse aromatase, a region
with high homology to the rat and human aromatase (38). Next
day, membranes were incubated for 2 hours at room temperature
with horseradish peroxidase conjugated goat antirabbit secondary antibody (Reference JAC-111– 035-003; Batch number
93362; Jackson Immuno Research), diluted 1:10 000 in Trisbuffered saline, with 0.1% Tween 20 and 5% BSA. A monoclonal antibody against ␤-actin (Reference A5316; Batch number
122M4755; Clone AC-74; Sigma; diluted 1:4000) was used as
loading control. Specific proteins were visualized with enhanced
chemiluminescence detection reagent according to the manufacturer’s instructions (Amersham). Densitometry and quantification of the bands were carried out using the Quantity One software (Bio-Rad).
In the rat spinal cord the aromatase antibody resulted in the
staining of several bands (Figure 1), including a band at 51 KDa,
corresponding to the molecular weight of aromatase. This band
was eliminated by the preabsorption of the antiserum with the
immunizing peptide (Figure 1) and is the band that was quantified in the study. In addition, the preabsorption of the antiserum
resulted in the elimination of a band between 75–100 KDa. The
nature of this band is unknown, although it has been described
that aromatase forms oligomers in living cells (39, 40).
Aromatase inhibitor administration
To investigate the role of aromatase in the development of
central pain syndrome we use the aromatase specific inhibitor,
letrozole (Sigma). Letrozole was intrathecally injected (1 mg/kg)
in a volume of 20 ␮L/250g in hydroxypropyl cellulose (0.3% in
water), 5 minutes after electrical lesion and daily for the next 28
days.
RT-PCR
Rats (eight rats for each group) were anesthetized with ketamine (60 mg/kg) and xylazine (20 mg/kg) and killed by decapitation at different time intervals after surgery. Thoracic spinal
segments 8 –10 of the spinal cord were rapidly removed. The
dorsal half of the spinal cord segments was dissected, frozen on
dry ice, and stored at ⫺80°C. Total RNA from snap-frozen spinal cord was extracted using Trizol reagent according to the
manufacturer’s protocol (Invitrogen). RNA was eluted in 50 ␮l
of RNase-free water and absorbance was measured at 260 nm to
determine concentrations. cDNA was synthesized from 2 ␮g of
total RNA by using a high-capacity cDNA reverse transcription
kit (Applied Biosystems). After reverse transcription, the cDNA
was diluted 1:20 and 5 ␮l were amplified by real-time PCR using
SYBR Green master mix (Applied Biosystems) in an ABI Prism
7500 Sequence Detector (Applied Biosystems), with conventional Applied Biosystems cycling parameters (40 cycles of 95°C,
15 seconds; 60°C, 1 minute). 18S 18S rRNA was selected as the
control housekeeping gene. Primer sequences were designed using Primer Express (Applied Biosystems) and were as follows:
Aromatase, forward:
5⬘-TCCCATGGCAGATTCTTGTG-3⬘
And reverse:
5⬘-TCTCCTCTCCACTGATCCAGACT-3⬘
ER␣, forward:
5⬘-ACCAATGCACCATCGATAAGAA-3⬘
And reverse:
5⬘-TCTTTTCGTATCCCGCCTTTC-3⬘
ER␤, forward:
5⬘-TGGGTATCATTACGGCGTTTG-3⬘
And reverse:
5⬘-CTGATTCGTGGCTGGACAGA-3⬘
18S rRNA, forward:
5⬘-AGACGAACCAGAGCGAAAGC-3⬘
And reverse:
5⬘-TGGTCGGAACTACGACGGTAT-3⬘
Values were normalized to 18S rRNA values and the ⌬⌬CT
method was used to determine relative expression levels. Statistics were performed using ⌬⌬CT values.
Behavioral assessment
Behavioral assessments including motor activity and mechanical allodynia were investigated pre- and postsurgery. To determine the effects of STT lesion and letrozole treatment over time,
we applied the behavioral tests to all animals on days 3, 7, 14, 21,
and 28 after injury. All the behavioral tests were performed on
the rats used for immunohistochemistry.
Open field test
Motor activity was assessed using the open field test apparatus, which consisted of a 60 cm ⫻ 60 cm ⫻ 40 cm black wooden
box. Animals were habituated in the room for 30 minutes and
then each of them was placed in one corner of the box and allowed to explore it freely for 5 minutes. For each trial, the open
field box was thoroughly cleaned with 70% ethanol solution and
4344
Ghorbanpoor et al
Aromatase Control Gliosis and Pain
Endocrinology, November 2014, 155(11):4341– 4355
Immunohistochemistry for ionized calcium-binding
adaper molecule 1 (Iba1) and glial fibrillary acidic
protein
Figure 1. Dorsal horn spinal cord tissue expresses aromatase (Aro). A,
Representative Western blots showing the expression of aromatase in
a sham-laminectomy and a STT-injury animal (laminectomized animal
with spinothalamic tract lesion) killed at day 28 (D28) after injury. A
band corresponding to the expect molecular weights of aromatase is
detected as a 51 kDa. B, Normal pattern of expression of Aro in spinal
cord tissue. C–F, Preincubation of the primary antibody with increasing
concentrations of the immunogenic peptide (C, 50 ng/ml; D, 100 ng/
ml; E, 500 ng/ml; and F, 1 mg/ml) resulted in the absence of the 51
kDa band. MW, molecular weight markers.
afterwards by a dry cloth. The experiments were conducted under artificial laboratory illumination (fluorescent lamps, above
level of box). The sessions were recorded by a camera positioned
right above the box hanging from the ceiling. Data were obtained
using Ethovision software (version 7), a video tracking system
for automation of behavioral experiments (Noldus Information
Technology, Netherlands). Distances traveled were recorded for
5 minutes as a behavior in open field box.
The activation of astrocytes and microglia after STT lesion
and letrozole administration was examined at different time intervals by immunohistochemistry. Animals were terminally
anesthetized with ketamine (65 mg/kg ip), and perfused through
the left cardiac ventricle, first with prewarmed (37°C) 0.1M
phosphate buffer saline (PBS) and then with 200 ml of 4% paraformaldehyde in 0.1M phosphate buffer (pH, 7.4). After perfusion, spinal cord segments (T8 –10) were removed and post fixed
in the same fixative overnight and washed three times with 0.1M
phosphate buffer (pH, 7.4). Samples were then sunk in 30%
sucrose-PBS and frozen in dry ice. Coronal sections of the spinal
cords, 50-␮m thick, were obtained using a sliding microtome
(HM 450, Microm International GmbH). Sections were stored in
ethylene-glycol based cryoprotectant at ⫺20°C until processing
for immunohistochemistry.
Immunohistochemistry was carried out in free-floating sections under moderate shaking. All washes and incubations were
performed in 0.1M phosphate buffer (pH, 7.4), containing 0.3%
BSA and 0.3% Triton X-100. To decrease variability, sections
for all experimental groups were processed in parallel in each
assay run. Endogenous peroxidase activity was quenched for 10
minutes at room temperature in a solution of 3% hydrogen peroxide in 30% methanol. Sections were incubated overnight at
4°C with either a rabbit polyclonal antibody against glial fibrillary acidic protein glial fibrillary acidic protein a marker of astroglia (Reference Z-0334, Batch number 00073719; Dako; diluted 1:1000), or a rabbit polyclonal antibody to Iba1 (Ionized
calcium binding adaptor molecule 1), a marker of microglia (Reference 019 –19741, Batch number WEK6254; Wako diluted
1:2000). Sections were rinsed in buffer and incubated for 2 hours
at room temperature with biotinylated goat antirabbit Ig G (Reference 31820, Batch number NH1599519; diluted 1:300). After
several washes in buffer, sections were incubated for 90 minutes
at room temperature with avidin-biotin-peroxidase complex
(ImmunoPure ABC peroxidase staining kit, Pierce). The reaction
product was revealed by incubating the sections with 2 ␮g/ml
3,3⬘-diaminobenzidine (SigmaAldrich) and 0.01% hydrogen
peroxide in 0.1M phosphate buffer. Finally, sections were dehydrated, mounted on gelatinized slides, coverslipped in Depex
mounting medium, and examined with a Leitz Laborlux microscope (Leica Microsystems).
Morphometric analysis
Mechanical allodynia
Mechanical allodynia was assessed as originally described by
Ren (41). Animals were habituated in the new cage for 10 minutes, then mechanical hind paw thresholds were measured bilaterally using calibrated von Frey filaments (Stoelting). Filaments were applied to the dorsal surface of the paws based on
studies demonstrating that the dorsal approach more reliably
and consistently detects threshold changes. Stimulus response
function curves were prepared by plotting the paw withdrawal
threshold vs days postsurgery. Paw withdrawal threshold was
defined as the force at which the animal withdrew the paw to
three of the five stimuli delivered. Each animal was used as its
own control then pre- and postlesion responses were measured.
The morphometric analysis was performed on coded sections
by an investigator who was blind to the experimental groups.
Sections from six animals for each experimental group were analyzed. Iba1 and glial fibrillary acidic protein immunoreactive
cells were assessed in the dorsal horn contralateral and ipsilateral
to the STT injury, at T8 –T9 spinal cord level. The number of Iba1
and glial fibrillary acidic protein immunoreactive cells was estimated by the optical disector method (42), using total thickness
for disector height (43) and a counting frame of 55 ⫻ 55 ␮m. A
total of 32 counting frames were assessed per animal. The first
disector was placed in the lateral border of lamina III and then
four consecutive but not overlapping disectors were placed in
lamina III toward the medial border of the dorsal horn. Then,
three contiguous but not overlapping disectors were placed dor-
doi: 10.1210/en.2014-1158
sally to the previous ones. The procedure was repeated in four
sections for each animal. The results were averaged for each
animal and the value of each animal was used for the statistical
analysis. Section thickness was measured using a digital length
gauge device (Heidenhain-Metro MT 12/ND221) attached to
the stage of a Leitz microscope. All Iba1 or glial fibrillary acidic
protein immunoreactive cell somas that came into focus while
focusing down through the disector height and were inside the
disector frame were counted.
The proportion of Iba1-immunoreactive cells with different
morphologies was also assessed. Iba1-immunoreactive cells with
few cellular processes (two or fewer), cells showing four short
branches and also cells with numerous cell processes and a small
cell body were classified as resting microglia (44, 45). Iba1-immunoreactive cells with large somas and retracted and thicker
processes and cells with amoeboid cell body, numerous short
processes, and intense Iba1 immunostaining, were classified as
reactive microglia (44, 45). Approximately 100 Iba1-immunoreactive cells were assessed per animal.
Statistical analysis
Data were analyzed using a 2-way ANOVA, with treatment
and time after treatment as factors. Kolmogorov-Smirnov test
provided with SPSS program (SPSS) was used to assess the assumption of normality. Data were not always normally distributed. Therefore, to satisfy the assumption of normality for the
ANOVA, data were transformed when necessary by the natural
logarithm transformation. Assessing the assumption of normality was performed after each transformation using the Kolmogorov-Smirnov test. For data that were homoscedastic and normally distributed, Bonferroni’s test was used for post hoc
comparisons. For those data that were heteroscedastic and normally distributed, Games-Howell’s test was used. If transformed
data were not normally distributed, nonparametric tests were
used (Kruskal-Wallis and post hoc pair-wise comparisons with
Mann-Whitney U test). When appropriate, 2-way ANOVAs
were followed by 1-way ANOVA split by the independent factor
(time) to further analyze the data. Pearson test was computed to
assess the possible correlation between two variables. Statistical
analyses were carried out with the SPSS 21.0 software package
(SPSS). The level of significance was set at P ⬍ .05. Data are
presented as mean ⫾ SEM from six animals per experimental
group and day of analysis, except for data of Figure 2, in which
N ⫽ 8.
Results
STT injury increases the mRNA and protein levels
of aromatase in the dorsal horn of the spinal cord
The mRNA and protein levels of aromatase were assessed in the dorsal horn of the spinal cord (T8 –T10 segments) of the animals from the sham-laminectomy and
STT groups at 3, 7, 14, 21, and 28 days after surgery.
Two-way ANOVA revealed a significant effect of surgery
(F1,70 ⫽ 317.595; P ⬍ .05) and time after surgery (F4,70 ⫽
5.079; P ⬍ .05) as well as a significant surgery ⫻ time
interaction (F4,70 ⫽ 3.777; P ⬍ .05) on aromatase mRNA
endo.endojournals.org
4345
levels. Post hoc comparisons showed that sham-laminectomy did not significantly affect the mRNA levels of aromatase (Figure 2A). Thus, similar mRNA levels of aromatase were detected from day 3–28 after surgery in shamlaminectomy animals. In contrast, in the STT-injured
animals, aromatase mRNA levels were significantly increased (P ⬍ .001) vs sham-laminectomy values from day
3–28 after injury (Figure 2A). In addition, aromatase
mRNA levels increased gradually from day 7–28 after STT
injury. At 28 days after STT injury, aromatase mRNA
levels in this experimental group were significantly increased compared with days 3 and 7 post STT injury (Figure 2A).
Two-way ANOVA revealed a significant effect of surgery (F1,50 ⫽ 65.248; P ⬍ .05) and time after surgery (F4,50
⫽ 2.905; P ⬍ .05) as well as a significant surgery ⫻ time
interaction (F4,50 ⫽ 2.534; P ⬍ .05) on aromatase protein
levels. As shown in figure 2B, post hoc comparisons revealed that STT injury induced an increase in aromatase
expression at days 21 and 28 post injury (P ⬍ .001).
STT injury increases the mRNA and protein levels
of ER␣ in the dorsal horn of the spinal cord
The mRNA and protein levels of ER␣ were assessed in
the dorsal horn of the spinal cord (T8 –T10 segments) of
the animals from the sham-laminectomy and STT-injury
groups at 3, 7, 14, 21, and 28 days after surgery. Two-way
ANOVA revealed a significant effect of surgery (F1,70 ⫽
41.579; P ⬍ .05) and time (F4,70 ⫽ 10.944; P ⬍ .05) as well
as a significant surgery ⫻ time interaction (F4,70 ⫽ 10.727;
P ⬍ .05) on ER␣ mRNA levels. Post hoc comparisons
revealed that sham-laminectomy did not significantly affect the mRNA levels of ER␣ (Figure 2C). In contrast, ER␣
mRNA levels were significantly increased in days 14 –28
after STT injury compared with sham-laminectomy values
and to the values in days 3 and 7 after STT injury (Figure
2C).
Two-way ANOVA revealed a significant effect of surgery (F1,50 ⫽ 6.846; P ⬍ .05) and time (F4,50 ⫽ 8.188; P ⬍
.05) as well as a significant surgery ⫻ time interaction
(F4,50 ⫽ 8.636; P ⬍ .05) on ER␣ protein levels. The post
hoc comparisons showed a significant increment in ER␣
expression at days 21 and 28 after STT injury (P ⬍ .05;
Figure 2D).
STT injury increases the mRNA and protein levels
of ER␤ in the dorsal horn of the spinal cord
The mRNA and protein levels of ER␤ were assessed in
the dorsal horn of the spinal cord (T8 –T10 segments) of
the animals from the sham-laminectomy and STT-injury
groups at 3, 7, 14, 21, and 28 days after surgery. Two-way
4346
Ghorbanpoor et al
Aromatase Control Gliosis and Pain
Endocrinology, November 2014, 155(11):4341– 4355
Figure 2. Quantitative analysis of A, aromatase; C, estrogen receptor ␣ (ER␣); and E, ER␤ mRNA, obtained by real-time PCR and examples of
Western blots and results of the densitometric analysis of B, aromatase (Aro); D, ER␣; and F, estrogen receptor ␤ (ER␤) protein levels in the dorsal
horn of the rat spinal cord. Quantitative analysis of mRNAs and proteins levels was examined at different days (D3–D28) after surgery. Protein
levels were normalized to ␤-actin. Sham-laminectomy, laminectomized animals; STT-injury, laminectomized animals with spinothalamic tract
lesion. For mRNA assessment the number of animals was eight and for protein analysis the number of animals was six in all experimental groups.
Data are mean ⫾ SEM. *, **, ***, significant differences vs sham group (*, P ⬍ .05; **, P ⬍ .01; ***, P ⬍ .001).
ANOVA revealed a significant effect of surgery (F1,70 ⫽
4.138; P ⬍ .05) and time (F4,70 ⫽ 11.567; P ⬍ .05) as
well as a significant surgery ⫻ time interaction (F4,70 ⫽
10.736; P ⬍ .05) on ER␤ mRNA levels. The post hoc
comparisons showed that sham-laminectomy did not
significantly affect the mRNA levels of ER␤ (Figure 2E).
In contrast, ER␤ mRNA levels were significantly increased in days 7 and 14 after STT injury compared with
sham-laminectomy levels (Figure 2E). After day 14,
ER␤ mRNA levels in STT-injury animals were decreased, reaching values less than control levels by day
28 after STT injury (Figure 2E).
Two-way ANOVA revealed a significant effect of surgery (F1,50 ⫽ 5.985; P ⬍ .05) and time (F4,50 ⫽ 2.834; P ⬍
.05) as well as a significant surgery ⫻ time interaction
(F4,50 ⫽ 3.736; P ⬍ .05) on ER␤ protein levels. Post hoc
comparisons revealed that STT injury induced an increase
in ER␤ expression at day 7 post injury (P ⬍ .01; Figure 2F).
Then, the levels of ER␤ returned to control values.
Aromatase inhibition enhances the effect of STT
injury on mechanical allodynia
Behavioral effects of aromatase inhibition were assessed at 3, 7, 14, 21, and 28 days after surgery. Behavioral
doi: 10.1210/en.2014-1158
data were not normally distributed. Therefore, data were
analyzed using the Kruskal-Wallis test. No significant differences were detected between the sham-laminectomyvehicle, STT-vehicle, and STT-letrozole groups in locomotor function using the open field test (Figure 3A).
Regarding mechanical pain (Figure 3, B and C),
Kruskal-Wallis test revealed a significant decrease in paw
withdrawal threshold after surgery in both hind paws of
rats (DF ⫽ 15; N ⫽ 108; P ⬍ .001). In all the experimental
Figure 3. Effect of unilateral electrolytic lesion of spinothalamic tract
on motor performance of animals and hind paw withdrawal threshold
at different days (D3–D28) after surgery. A, Motor activity; B,
contralateral hind paw withdrawal threshold; C, ipsilateral hind paw
withdrawal threshold. Sham-laminectomy-vehicle, laminectomized
animals with intrathecal injection of vehicle; STT-vehicle,
laminectomized animals with spinothalamic tract lesion and intrathecal
injection of vehicle; STT-letrozole, laminectomized animals with
spinothalamic tract lesion and intrathecal injection of the aromatase
inhibitor letrozole. The number of animals was six in all experimental
groups. Data are mean ⫾ SEM. &, Significant differences (P ⬍ .05)
between sham-laminectomy-vehicle and STT-vehicle group. *,
Significant differences (P ⬍ .05) between sham-laminectomy-vehicle
and STT-letrozole group. #, Significant differences (P ⬍ .05) between
STT-vehicle and STT-letrozole group.
endo.endojournals.org
4347
groups the withdrawal threshold was decreased in both
hind paws at days 3 after surgery. Then the withdrawal
threshold showed a different pattern in each experimental
group. In sham-laminectomy-vehicle animals the withdrawal threshold showed a progressive increase from day
7–28 after surgery, reaching by day 14 values in both hind
paws that were not significantly different from control
levels (day 0).
In the STT-vehicle and STT-letrozole animals the withdrawal threshold continued to decrease until day 7 after
surgery in both hind paws. Subsequent post hoc MannWhitney U tests revealed that there was a significant difference in withdrawal threshold in contralateral and ipsilateral hind paws between STT-vehicle and the shamlaminectomy-vehicle group at day 7 (n ⫽ 6, P ⫽ .005, and
P ⫽ .02, respectively). Then the withdrawal threshold
showed a moderate increase on day 14 in the STT-vehicle
animals to values not different from sham-laminectomyvehicle levels. Then, the paw withdrawal threshold levels
in STT-vehicle animals reached a plateau from day 14 –28
after surgery, but never recovered to control (day 0) values
and remained significantly decreased compared with
sham-laminectomy-vehicle values on days 21 (n ⫽ 6; P ⫽
.012) and 28 (n ⫽ 6; P ⫽ .012) after surgery (Figure 3, B
and C).
In the STT-letrozole animals, the withdrawal threshold
continued to progressively decrease after day 7 and did not
show the plateau from day 14 –28 that was observed in the
in the STT-vehicle animals. Thus, the STT-letrozole animals showed a significant decrease in the withdrawal
threshold in both the contralateral and ipsilateral hind
paws at days 14 and 28 after lesion compared with STTvehicle animals (n ⫽ 6; day 14, contralateral P ⫽ .020; day
14 ipsilateral P ⫽ .020; day 28 contralateral P ⫽ .006; day
28, ipsilateral P ⫽ .004). In addition, STT-letrozole animals showed a significant decrease in the withdrawal
threshold in both the contralateral and ipsilateral hind
paws from day 7 (n ⫽ 6; P ⫽ .024 and P ⫽ .006, respectively) to day 28 (n ⫽ 6; P ⫽ .004 and P ⫽ .004, respectively) after lesion compared with sham-laminectomy-vehicle animals (Figure 3, B and C).
Aromatase inhibition enhances the effect of STT
injury on microglial activation
Immunoreactivity for the microglia cell marker Iba1
was assessed in the dorsal horn, contralateral and ipsilateral to the STT injury, at T8-T9 spinal cord level. Some
neuroinflammatory conditions result in the proliferation
of microglia. However, other neuroinflammatory conditions affect the activation of microglia without changes in
the number of microglia cells (46). Therefore, two parameters were analyzed in the present study: the total number
4348
Ghorbanpoor et al
Aromatase Control Gliosis and Pain
Endocrinology, November 2014, 155(11):4341– 4355
Figure 4. Effects of unilateral lesion of spinothalamic tract on microglial reactivity in the dorsal
horn contralateral to the STT electrolytic lesion. The upper panels show representative examples
of Iba1-immunoreactive microglia cells in the dorsal horn at different days (D3–D28) after
surgery. The bottom panel shows the results of the quantitative analysis of the proportion of
Iba1-positive microglia cells with reactive phenotype and a schematic drawing indicating the
dorsal horn evaluated and the site of the lesion in the ventrolateral white matter. Shamlaminectomy-vehicle, laminectomized animals with intrathecal injection of vehicle; STT-vehicle,
laminectomized animals with spinothalamic tract lesion and intrathecal injection of vehicle; STTletrozole, laminectomized animals with spinothalamic tract lesion and intrathecal injection of the
aromatase inhibitor letrozole. The number of animals was six in all experimental groups. Data are
mean ⫾ SEM. *, ***, Significant differences (*, P ⬍ .05; ***, P ⬍ .001) vs the shamlaminectomy-vehicle group. #, ##, Significant differences (#, P ⬍ .05; ##, P ⬍ .01) vs the STTvehicle group.
of microglia cells and the proportion
of microglia cells with reactive phenotype. Examples of Iba1-immunoreactive microglia in the contralateral and ipsilateral dorsal horns are
shown in Figures 4 and 5, respectively. There were no significant effects of treatment and time after
treatment on the total number of
Iba1-immunoreactive cells (data not
shown). In contrast, in the contralateral dorsal horn, two-way ANOVA
revealed a significant effect of
treatment, time, and treatment ⫻
time interaction on the percentage
of microglia cells with reactive
phenotype (treatment: F2,75 ⫽
151.082; P ⬍ .05; Time after treatment: F4,75 ⫽ 10.551; P ⬍ .05;
treatment ⫻ time interaction: F8,75
⫽ 3.794; P ⬍ .05).
In the ipsilateral dorsal horn, twoway ANOVA revealed a significant
effect of treatment and time after
treatment on the percentage of microglia cells with reactive phenotype
(treatment: F2,75 ⫽ 88.726; P ⬍ .05;
Time after treatment: F4,75 ⫽ 3.658;
P ⬍ .05). However, there was not a
significant treatment ⫻ time interaction (F8,75 ⫽ 1.725; P ⬍ .106).
Post hoc comparisons revealed
that the STT-vehicle injury induced
an increase in the reactive morphology of microglial cells from day 3–28
post lesion in both the contralateral
(P ⬍ .05 on day 3 and P ⬍ .001 on
days 7–28) and ipsilateral dorsal
horns (P ⬍ .05 on day 3 and day 21
and P ⬍ .01 on day days 7–28).
The treatment with the aromatase
inhibitor enhanced the reactive microgliosis induced by the lesion (Figures 4 and 5). Thus, the proportion
of microglia cells with reactive phenotype was increased on days 3, 21,
and 28 after STT-letrozole in the
contralateral dorsal horn compared
with STT-vehicle animals (P ⬍ .05).
In addition, the proportion of microglia cells with reactive phenotype
was increased from day 7–28 after
doi: 10.1210/en.2014-1158
endo.endojournals.org
4349
STT-letrozole in the ipsilateral dorsal horn compared with STT-vehicle
animals (P ⬍ .05 and P ⬍ .01).
Figure 5. Effects of unilateral lesion of spinothalamic tract on microglial reactivity in the dorsal
horn ipsilateral to the STT electrolytic lesion. The upper panels show representative examples of
Iba1-immunoreactive microglia cells in the dorsal horn at different days (D3–D28) after surgery.
The bottom panel shows the results of the quantitative analysis of the proportion of Iba1-positive
microglia cells with reactive phenotype and a schematic drawing indicating the dorsal horn
evaluated and the site of the lesion in the ventrolateral white matter. Sham-laminectomy-vehicle,
laminectomized animals with intrathecal injection of vehicle; STT-vehicle, laminectomized animals
with spinothalamic tract lesion and intrathecal injection of vehicle; STT-letrozole, laminectomized
animals with spinothalamic tract lesion and intrathecal injection of the aromatase inhibitor
letrozole. The number of animals was six in all experimental groups. Data are mean ⫾ SEM. *,
**, ***, Significant differences (*, P ⬍ .05; **, P ⬍ .01; ***, P ⬍ .001) vs the shamlaminectomy-vehicle group. #, ##, ###, Significant differences (#, P ⬍ .05; ##, P ⬍ .01; ###, P ⬍
.001) vs the STT-vehicle group.
Aromatase inhibition enhances
the effect of STT injury on glial
fibrillary acidic protein
immunoreactivity
Examples of glial fibrillary acidic
protein-immunoreactive astrocytes
in the contralateral and ipsilateral
dorsal horns are shown in Figures 6
and 7, respectively. Two-way
ANOVA revealed a significant effect
of the treatment on the number of
glial fibrillary acidic protein immunoreactive cells in both the contralateral and the ipsilateral dorsal horns
(F2,75 ⫽ 54.609; P ⬍ .05, contralateral and F2,75 ⫽ 20.868; P ⬍ .05,
ipsilateral). There was not a significant effect of time (F4,75 ⫽ 1.043;
P ⫽ .391, contralateral; and F4,75 ⫽
1.293; P ⫽ .280, ipsilateral) and
there was not a significant treatment ⫻ time interaction (F8,75 ⫽
1.195; P ⫽ .314, contralateral; and
F8,75 ⫽ 0.792; P ⫽ .611, ipsilateral)
on the number of glial fibrillary
acidic protein immunoreactive
cells.
Post hoc comparisons revealed
that STT-vehicle resulted in a significant increase in the number of glial
fibrillary acidic protein immunoreactive astrocytes on days 14 –28 (P ⬍
.05) in both the contralateral and the
ipsilateral dorsal horns compared
with control animals.
The treatment with the aromatase
inhibitor enhanced the effect of STT
injury on the number of glial fibrillary acidic protein immunoreactive
cells in the dorsal horn (Figures 6 and
7). Thus, the number of glial fibrillary acidic protein immunoreactive
cells was significantly increased in
STT-letrozole animals compared
with STT-vehicle animals on days
14 –28 (P ⬍ .01) in the contralateral
side and on days 14 –28 in the ipsilateral side (P ⬍ .05; Figures 6 and 7).
4350
Ghorbanpoor et al
Aromatase Control Gliosis and Pain
Endocrinology, November 2014, 155(11):4341– 4355
Figure 6. Effects of unilateral lesion of spinothalamic tract on glial fibrillary acidic protein
immunoreactive astrocytes in the dorsal horn contralateral to the STT electrolytic lesion. The
upper panels show representative examples of glial fibrillary acidic protein-immunoreactive cells
in the dorsal horn at different days (D3–D28) after surgery. The bottom panel shows the results
of the quantitative analysis of the number of glial fibrillary acidic protein-immunoreactive cells
and a schematic drawing indicating the dorsal horn evaluated and the site of the lesion in the
ventrolateral white matter. Sham-laminectomy-vehicle, laminectomized animals with intrathecal
injection of vehicle; STT-vehicle, laminectomized animals with spinothalamic tract lesion and
intrathecal injection of vehicle; STT-letrozole, laminectomized animals with spinothalamic tract
lesion and intrathecal injection of the aromatase inhibitor letrozole. The number of animals was
six in all experimental groups. Data are mean ⫾ SEM. *, **, ***, Significant differences (*, P ⬍
.05; **, P ⬍ .01; ***, P ⬍ .001) vs the sham-laminectomy-vehicle group. ##, ###, Significant
differences (##, P ⬍ .01; ###, P ⬍ .001) vs the STT-vehicle group.
The proportion of reactive
microglia and the number of
glial fibrillary acidic protein
immunoreactive cells in the
dorsal horn were significantly
and positively correlated with
mechanical allodynia in both
hind paws.
To determine whether the amount
of gliosis in the dorsal horn correlates
with the levels of mechanical allodynia
we performed a correlation analysis of
the paw withdrawal threshold and
dorsal horn gliosis (proportion of Iba1
microglia cells with reactive phenotype or number of glial fibrillary acidic
protein immunoreactive cells) in the
animals killed 28 days after surgery,
using a pool of the three experimental
groups (N ⫽ 18, Figure 8). The results
suggest a significant and negative correlation between the paw withdrawal
threshold and reactive gliosis. This
correlation was significant for both
hind paws and for both the contralateral and ipsilateral dorsal horns (Figure 8). To determine whether the correlation is the result of individual or
group differences we calculated the
same correlation in each groups separately. Even with the small number
(n ⫽ 6) some correlations remained
significant: allodynia contralateral/reactive microglia (r2 ⫽ 0.84; P ⫽ .0095)
in the STT-vehicle group; allodynia ipsilateral/reactive microglia (r2 ⫽ 0.77;
P ⫽ .0216) in the STT-letrozole group;
allodynia contralateral/glial fibrillary
acidic protein–positive cells (r2 ⫽
0.92; P ⫽ .0023) in the STT-letrozole
group and allodynia ipsilateral/glial
fibrillary acidic protein–positive cells
(r2 ⫽ 0.77; P ⫽ .0207) in the STTletrozole group. This suggests that individual differences contribute to the
correlations.
Discussion
Our findings suggest that STT injury
enhances the mRNA and protein lev-
doi: 10.1210/en.2014-1158
Figure 7. Effects of unilateral lesion of spinothalamic tract on glial fibrillary acidic protein
immunoreactive astrocytes in the dorsal horn ipsilateral to the STT electrolytic lesion. The upper
panels show representative examples of glial fibrillary acidic protein-immunoreactive cells in the
dorsal horn at different days (D3–D28) after surgery. The bottom panel shows the results of the
quantitative analysis of the number of glial fibrillary acidic protein-immunoreactive cells and a
schematic drawing indicating the dorsal horn evaluated and the site of the lesion in the
ventrolateral white matter. Sham-laminectomy-vehicle, laminectomized animals with intrathecal
injection of vehicle; STT-vehicle, laminectomized animals with spinothalamic tract lesion and
intrathecal injection of vehicle; STT-letrozole, laminectomized animals with spinothalamic tract
lesion and intrathecal injection of the aromatase inhibitor letrozole. The number of animals was
six in all experimental groups. Data are mean ⫾ SEM. *, **, ***, Significant differences (*, P ⬍
.05; **, P ⬍ .01; ***, P ⬍ .001) vs the sham-laminectomy-vehicle group. #, ##, ###, Significant
differences (#, P ⬍ .05; ##, P ⬍ .01; ###, P ⬍ .001) vs the STT-vehicle group.
endo.endojournals.org
4351
els of aromatase and ERs in the dorsal horn of the rat spinal cord. These
findings are in agreement with previous studies that have shown that
different forms of injury in the CNS
result in the induction of the expression of aromatase (21–23) and ERs
in the neural tissue (47– 49). The increase in the expression of aromatase
and ERs after STT lesion suggests
that this form of injury may increase
local estradiol levels and local estradiol signaling in the dorsal horn.
A transient increase in the expression of ER␤ at day 7 after STT injury
was followed by an increase in the
expression of ER␣ and aromatase on
days 21 and 28 after STT injury. The
sequential increase in the expression
of ER␤ followed by ER␣ suggests a
different function for both receptors
at different time points after injury.
The different function of ERs may be
related with the different cell types
activated after the axotomy of the
neurons of origin of STT in the dorsal horn by the contralateral STT injury. Axotomy results in the sequential activation of neurons, microglia,
astrocytes, and oligodendrocytes
(50) and all these cell types express
ERs in the spinal cord (51–54). The
onset of neuropathic pain also involves a sequential activation of microglia and astrocytes. Thus, the sequential activation of the expression
of ER␤ and ER␣ may be related with
the sequential activation of microglia, which expresses ER␤ in the spinal cord (54), and astrocytes, which
express predominantly ER␣ in the
spinal cord (52). In addition, the
changes in aromatase expression in
the dorsal horn paralleled the
changes in the expression of ER␣,
suggesting a possible role of locally
synthesized estradiol on this ER subtype after STT injury.
Increased estradiol synthesis and
signaling in the dorsal horn of the
spinal cord after STT injury may
contribute to regulate pain percep-
4352
Ghorbanpoor et al
Aromatase Control Gliosis and Pain
Endocrinology, November 2014, 155(11):4341– 4355
spinal cord reduces pain perception
in this model. Thus, we may postulate that the increased expression of
aromatase and ERs after STT may
represent an endogenous mechanism
to regulate pain by increasing local
estradiol levels and by enhancing estradiol-signaling mechanisms involved in the regulation of nociception in the spinal cord. Additional
studies should determine whether
aromatase inhibition affects pain
perception under basal conditions.
The absence of a sham group treated
with letrozole was a limitation in our
experimental design. Another limitation of the study is that we only
tested a single dose of letrozole and a
single route of administration. The
dose and route of administration
Figure 8. Correlation analysis of allodynia hind paw withdrawal threshold and the proportion of
was selected on the basis of a previIba-positive microglia cells with reactive phenotype and the number of glial fibrillary acidic
ous study showing that the intratheprotein immunoreactive cells in animals of all experimental groups and killed 28 days after
cal injection of 1 mg/kg letrozole
surgery. A, Correlation of allodynia withdrawal threshold for the hind paw contralateral to the
lesion and the proportion of reactive microglia in the dorsal horn contralateral to the lesion. B,
causes apoptosis in dorsal root ganCorrelation of allodynia withdrawal threshold for the hind paw ipsilateral to the lesion and the
glion neurons in rats submitted to
proportion of reactive microglia in the dorsal horn ipsilateral to the lesion. C, Correlation of
sciatic nerve chronic constriction inallodynia withdrawal threshold for the hind paw contralateral to the lesion and the number of
glial fibrillary acidic protein-immunoreactive cells in the dorsal horn contralateral to the lesion. D,
jury (57). However, it would be inCorrelation of allodynia withdrawal threshold for the hind paw ipsilateral to the lesion and the
teresting to know whether systemic
number of glial fibrillary acidic protein-immunoreactive cells in the dorsal horn ipsilateral to the
letrozole also enhances allodynia in
lesion.
our model. In women, 2.5 mg letrotion, given that estradiol is involved in the regulation of zole orally result in almost-complete inhibition of body
nociception acting directly on pain-processing neurons of aromatatization (58) and in rats the sc administration of
the spinal cord (33, 55) and by a mechanism involving letrozole (5 mg/kg) increases formalin-induced tonic pain
ER␣ (56). Previous studies have shown that aromatase (59).
expression after brain injury is neuroprotective (24 –29).
As in other forms of CNS injury, STT injury resulted in
Thus, the increased aromatase and ER expression in the a significant increase in the proportion of microglia cells
CNS after local injury has been interpreted as an endog- with reactive phenotype and in the number of glial fibrilenous protective mechanism, mediated by the activation lary acidic protein immunoreactive astrocytes in the dorsal
of estradiol-neuroprotective signaling. According to this horn. Both parameters are indicative of reactive glial acinterpretation, the increased expression of aromatase and tivation. As also observed in other injury models, the acERs detected in the spinal cord after STT injury may also tivation of microglia preceded the increase in the number
represent a neuroprotective mechanism that may contrib- of glial fibrillary acidic protein immunoreactive cells. Miute to reduce secondary damage causing long-term dys- croglia and astrocytes participate in the regulation of pain
function of pain perception. To test this hypothesis we signaling by the release of a variety of molecules including
decided in the present study to assess whether aromatase proinflammatory cytokines (60 – 67). Several studies sugmay also be protective in the STT model. To this aim, we gest that microglia are activated during the onset and early
treated the animals with letrozole, an aromatase inhibitor, stages of neuropathic pain by neurotransmitters and neuand we assessed the effect of aromatase inhibition on me- romodulators released by neurons (65, 68). In turn, michanical allodynia.
croglia signals regulate the activity of pain processing neuOur findings suggest that 1 mg/kg intrathecal letrozole rons in the dorsal horn (65, 68, 69). Astrocytes are also
enhances the effect of STT on mechanical allodynia. This activated by microglia at latter stages and also contribute
finding suggests that local aromatase production in the to pain signaling (65, 68, 69).
doi: 10.1210/en.2014-1158
Estradiol is known to regulate the activation of astrocytes and microglia after different forms of CNS injury or
under conditions of neuroinflammation (70, 71). Thus,
given the role of glial cells in chronic pain (69) we hypothesized that at least part of the enhancing effect of aromatase inhibition on allodynia could be the result of an increased reactive gliosis. The intrathecal administration of
the aromatase inhibitor resulted in a significant increase in
the proportion of microglia cells with reactive phenotype
and in a significant increase in the number of glial fibrillary
acidic protein immunoreactive astrocytes after STT injury
compared with STT-injured animals injected with vehicle.
These findings, showing that the local inhibition of aromatase results in an increase in the gliotic response to STT
injury suggest that local estradiol production in the spinal
cord after STT is involved in the regulation of gliosis.
Therefore, the increased expression of aromatase in the
dorsal horn after STT injury may represent an endogenous
mechanism to control reactive gliosis, which in turn may
participate in the control of pain sensitivity.
In agreement with previous studies (7), the effect of STT
injury on mechanical allodynia was observed in both the
left and the right hind paws although the STT injury was
unilateral, in the right side. The mechanisms involved in
the bilateral propagation of altered pain sensitivity are
unclear. Among other possibilities it has been proposed
that reactive astrocytes may spread excitation and neuroinflammation from one side to the other of the spinal cord
gray matter through gap junctions (72). Interestingly, in
our study gliosis was observed in both dorsal horns. Furthermore, the amount of gliosis in each dorsal horn positively correlated with mechanical allodynia in both hind
paws. This finding strongly supports the implication of
gliosis on allodynia. Therefore, our findings suggest that
the induction of aromatase expression in the spinal cord
after STT injury decreases mechanical allodynia by decreasing reactive gliosis in the dorsal horn.
Acknowledgments
We thank Ms Ana Belen Lopez-Rodriguez, Ms Isabel Ruiz-Palmero and Ms Estefania Acaz-Fonseca for their help with the immunohistochemical and RT-PCR procedures. The authors also
appreciate the Research Council of the University of Tehran for
their valuable patronage.
Address all correspondence and requests for reprints to: Luis
M. Garcia-Segura, Instituto Cajal, CSIC, Avenida Doctor Arce
37, E-28002 Madrid, Spain. E-mail: [email protected].
This work was supported by Ministerio de Economía y Competitividad, Spain (BFU2011–30217-C03– 01) and Neurobiol-
endo.endojournals.org
4353
ogy Research Center of Shahid Beheshti University of Medical
Sciences Research Funds.
Disclosure Summary: The authors have nothing to disclose.
References
1. Treede RD, Jensen TS, Campbell JN,. Neuropathic pain: Redefinition and a grading system for clinical and research purposes. Neurology. 2008;70(18):1630 –1635.
2. Sandkühler R. Models and mechanisms of hyperalgesia and allodynia. Physiol Rev. 2009;89(2):707–758.
3. Baron R, Binder A, Wasner G. Neuropathic pain: Diagnosis, pathophysiological mechanisms, and treatment. Lancet Neurol. 2010;
9(8):807– 819.
4. Von Hehn CA, Baron R, Woolf CJ. Deconstructing the neuropathic
pain phenotype to reveal neural mechanisms. Neuron. 2012;73(4):
638 – 652.
5. Moore R, Derry S, Aldington D, Cole P, Wiffen P. Amitriptyline for
neuropathic pain and fibromyalgia in adults. Cochrane Database
Syst Rev. 2012;12(12):CD008242
6. Moore R, Wiffen P, Derry S, Toelle T, Rice A. Gabapentin for
chronic neuropathic pain and fibromyalgia in adults. Cochrane Database Syst Rev. 2014;27(4):CD007938.
7. Wang G, Thompson SM. Maladaptive homeostatic plasticity in a
rodent model of central pain syndrome: Thalamic hyperexcitability
after spinothalamic tract lesions. J Neurosci. 2008;28(46):11959 –
11969.
8. Masri R, Quiton RL, Lucas JM, Murray PD, Thompson SM, Keller
A. Zona incerta: A role in central pain. J Neurophysiol. 2009;102(1):
181–191.
9. McMahon SB, Malcangio M. Current challenges in glia-pain biology. Neuron. 2009;64(1):46 –54.
10. Cao H, Zhang YQ. Spinal glial activation contributes to pathological pain states. Neurosci Biobehav Rev. 2008;32(5):972–983.
11. Gao YJ, Ji RR. Chemokines, neuronal-glial interactions, and central
processing of neuropathic pain. Pharmacol Ther. 2010;126(1):56 –
68.
12. Naseri K, Saghaei E, Abbaszadeh F, et al. Role of Microglia and
Astrocyte in Central Pain Syndrome Following Electrolytic Lesion at
the Spinothalamic Tract in Rats. J Mol Neurosci. 2013;49(3):470 –
479.
13. Arevalo MA, Santos-Galindo M, Bellini MJ, Azcoitia I, GarciaSegura LM. Actions of estrogens on glial cells: Implications for neuroprotection. Biochim Biophys Acta. 2010;1800(10):1106 –1012.
14. Amandusson Å, Blomqvist A. Estrogenic influences in pain processing. Front Neuroendocrinol. 2013;34(4):329 –349.
15. Gintzler AR, Liu NJ. Importance of sex to pain and its amelioration;
relevance of spinal estrogens and its membrane receptors. Front
Neuroendocrinol. 2012;33(4):412– 424.
16. Saghaei E, Abbaszadeh F, Naseri K, et al. Estradiol attenuates spinal
cord injury-induced pain by suppressing microglial activation in
thalamic VPL nuclei of rats. Neurosci Res. 2013;75(4):316 –323.
17. Balthazart J, Ball GF. Is brain estradiol a hormone or a neurotransmitter? Trends Neurosci. 2006;29(5):241–149.
18. Fester L, Prange-Kiel J, Jarry H, Rune GM. Estrogen synthesis in the
hippocampus. Cell Tissue Res. 2011;345(3):285–294.
19. Saldanha CJ, Remage-Healey L, Schlinger BA. Synaptocrine signaling: Steroid synthesis and action at the synapse. Endocr Rev. 2011;
32(4):532–549.
20. Cornil CA, Seredynski AL, de Bournonville C, et al. Rapid control
of reproductive behaviour by locally synthesised oestrogens: Focus
on aromatase. J Neuroendocrinol. 2013;25(11):1070 –1078.
21. Garcia-Segura LM, Wozniak A, Azcoitia I, Rodriguez JR, Hutchison RE, Hutchison JB. Aromatase expression by astrocytes after
4354
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
Ghorbanpoor et al
Aromatase Control Gliosis and Pain
brain injury: Implications for local estrogen formation in brain repair. Neuroscience. 1999;89(2):567–578.
Peterson RS, Saldanha CJ, Schlinger BA. Rapid Upregulation of
Aromatase mRNA and Protein Following Neural Injury in the Zebra
Finch (Taeniopygia guttata). J Neuroendocrinol. 2001;13(4):317–
323.
Duncan KA, Saldanha CJ. Neuroinflammation induces glial aromatase expression in the uninjured songbird brain. J Neuroinflammation. 2011;8(1):81–92.
Azcoitia I, Sierra A, Veiga S, Honda S, Harada N, Garcia-Segura
LM. Brain aromatase is neuroprotective. J Neurobiol. 2001;47(4):
318 –329.
McCullough LD, Blizzard K, Simpson ER, Oz OK, Hurn PD. Aromatase cytochrome P450 and extragonadal estrogen play a role in
ischemic neuroprotection. J Neurosci. 2003;23(25):8701– 8705.
Garcia-Segura LM, Veiga S, Sierra A, Melcangi RC, Azcoitia I. Aromatase: A neuroprotective enzyme. Prog Neurobiol. 2003;71(1):
31– 41.
Wynne RD, Walters BJ, Bailey DJ, Saldanha CJ. Inhibition of injuryinduced glial aromatase reveals a wave of secondary degeneration in
the songbird brain. Glia. 2008;56(1):97–105.
Saldanha CJ, Duncan KA, Walters BJ. Neuroprotective actions of
brain aromatase. Front Neuroendocrinol. 2009;30(2):106 –118.
Duncan KA, Walters BJ, Saldanha CJ. Traumatized and inflamed–
but resilient: Glial aromatization and the avian brain. Horm Behav.
2013;63(2):208 –215.
Evrard HC. Estrogen synthesis in the spinal dorsal horn: A new
central mechanism for the hormonal regulation of pain. Am J
Physiol Regul Integr Comp Physiol. 2006;291(2):291–299.
Evrard HC, Balthazart J. Rapid regulation of pain by estrogens
synthesized in spinal dorsal horn neurons. J Neurosci. 2004;24(33):
7225–7229.
Liu NJ, Chakrabarti S, Schnell S, Wessendorf M, Gintzler AR. Spinal
synthesis of estrogen and concomitant signaling by membrane estrogen receptors regulate spinal ␬- and ␮-opioid receptor heterodimerization and female-specific spinal morphine antinociception. J Neurosci. 2011;31(33):11836 –11845.
Amandusson A, Hermanson O, Blomqvist A. Colocalization of oestrogen receptor immunoreactivity and preproenkephalin mRNA expression to neurons in the superficial laminae of the spinal and medullary dorsal horn of rats. Eur J Neurosci. 1996;8(11):2440 –2445.
Shughrue PJ, Lane MV, Merchenthaler I. Comparative Distribution
of Estrogen Receptor-alpha and -beta mRNA in the Rat Central
Nervous System. J Comp Neurol. 1997;388:507–525.
Papka RE, Storey-Workley M, Shughrue PJ, et al. Estrogen receptoralpha and beta- immunoreactivity and mRNA in neurons of sensory
and autonomic ganglia and spinal cord. Cell Tissue Res. 2001;
304(2):193–214.
Platania P, Laureanti F, Bellomo M, et al. Differential expression of
estrogen receptors alpha and beta in the spinal cord during postnatal
development: Localization in glial cells. Neuroendocrinology. 2003;
77(5):334 –340.
Obata H, Li X, Eisenach JC. Spinal adenosine receptor activation
reduces hypersensitivity after surgery by a different mechanism than
after nerve injury. Anesthesiology. 2004;100(5):1258 –1262.
Beyer C, Green SJ, Barker PJ, Huskisson NS, Hutchison JB. Aromatase-immunoreactivity is localised specifically in neurones in the
developing mouse hypothalamus and cortex. Brain Res. 1994;
638(1–2):203–210.
Praporski S, Ng SM, Nguyen AD, et al. Organization of cytochrome
P450 enzymes involved in sex steroid synthesis: Protein-protein interactions in lipid membranes. J Biol Chem. 2009;284(48):33224 –
33232.
Jiang W, Ghosh D. Motion and flexibility in human cytochrome
p450 aromatase. PLoS One. 2012;7(2):e32565.
Ren K. An Improved method for assessing mechanical allodynia in
the rat. Physiol Behav. 1999;67(5):711–716.
Endocrinology, November 2014, 155(11):4341– 4355
42. Howard CV, Reed MG. Unbiased stereology. Three-dimensional
measurement in microscopy. Oxford: Bios; 1998.
43. Hatton WJ, von Bartheld CS. Analysis of cell death in the trochlear
nucleus of the chick embryo: Calibration of the optical disector
counting method reveals systematic bias. J Comp Neurol. 1999;
409(2):169 –186.
44. Diz-Chaves Y, Pernía O, Carrero P, Garcia-Segura LM. Prenatal
stress causes alterations in the morphology of microglia and the
inflammatory response of the hippocampus of adult female mice.
J Neuroinflammation. 2012;9(1):71– 87.
45. Diz-Chaves Y, Astiz M, Bellini MJ, Garcia-Segura LM. Prenatal
stress increases the expression of proinflammatory cytokines and
exacerbates the inflammatory response to LPS in the hippocampal
formation of adult male mice. Brain Behav Immun. 2013;28:196 –
206.
46. Tapia-Gonzalez S, Carrero P, Pernia O, Garcia-Segura LM, DizChaves Y. Selective oestrogen receptor (ER) modulators reduce microglia reactivity in vivo after peripheral inflammation: Potential
role of microglial ERs. J Endocrinol. 2008;198(1):219 –230.
47. Blurton-Jones M, Tuszynski MH. Reactive astrocytes express estrogen receptors in the injured primate brain. J Comp Neurol. 2001;
433(1):115–123.
48. García-Ovejero D, Veiga S, García-Segura LM, Doncarlos LL. Glial
expression of estrogen and androgen receptors after rat brain injury.
J Comp Neurol. 2002;450(3):256 –271.
49. Dubal DB, Rau SW, Shughrue PJ, et al. Differential modulation of
estrogen receptors (ERs) in ischemic brain injury: A role for ER alpha
in estradiol-mediated protection against delayed cell death. Endocrinology. 2006;147(6):3076 –3084.
50. Aldskogius H, Kozlova EN. Central neuron-glial and glial-glial interactions following axon injury. Prog Neurobiol. 1998;55(1):1–
26.
51. Arvanitis DN, Wang H, Bagshaw RD, Callahan JW, Boggs JM.
Membrane-associated estrogen receptor and caveolin-1 are present
in central nervous system myelin and oligodendrocyte plasma membranes. J Neurosci Res. 2004;75(5):603– 613.
52. Giraud SN, Caron CM, Pham-Dinh D, Kitabgi P, Nicot AB. Estradiol inhibits ongoing autoimmune neuroinflammation and NF-␬Bdependent CCL2 expression in reactive astrocytes. Proc Natl Acad
Sci U S A. 2010;107(18):8416 – 8421.
53. Lee JY, Choi SY, Oh TH, Yune TY. 17␤-Estradiol inhibits apoptotic
cell death of oligodendrocytes by inhibiting RhoA-JNK3 activation
after spinal cord injury. Endocrinology. 2012;153(8):3815–3827.
54. Wu WF, Tan XJ, Dai Y, Krishnan V, Warner M, Gustafsson JÅ.
Targeting estrogen receptor ␤ in microglia and T cells to treat experimental autoimmune encephalomyelitis. Proc Natl Acad Sci U S
A. 2013;110(9):3543–3548.
55. Amandusson A, Hallbeck M, Hallbeck AL, Hermanson O,
Blomqvist A. Estrogen-induced alterations of spinal cord enkephalin gene expression. Pain. 1999;83(2):243–248.
56. Zhong YQ, Li KC, Zhang X. Potentiation of excitatory transmission
in substantia gelatinosa neurons of rat spinal cord by inhibition of
estrogen receptor alpha. Mol Pain. 2010;6(1):92–100.
57. Schaeffer V, Meyer L, Patte-Mensah C, Eckert A, Mensah-Nyagan
AG. Sciatic nerve injury induces apoptosis of dorsal root ganglion
satellite glial cells and selectively modifies neurosteroidogenesis in
sensory neurons. Glia. 2010;58(2):169 –180.
58. Geisler J, Haynes B, Anker G, Dowsett M, Lønning PE. Influence of
letrozole and anastrozole on total body aromatization and plasma
estrogen levels in postmenopausal breast cancer patients evaluated
in a randomized, cross-over study. J Clin Oncol. 2002;20(3):751–
757.
59. Moradi-Azani M, Ahmadiani A, Amini H. Increase in formalininduced tonic pain by 5alpha-reductase and aromatase inhibition in
female rats. Pharmacol Biochem Behav. 2011;98(1):62– 66.
60. Mika J, Zychowska M, Popiolek-Barczyk K, Rojewska E, Przew-
doi: 10.1210/en.2014-1158
61.
62.
63.
64.
65.
66.
locka B. Importance of glial activation in neuropathic pain. Eur
J Pharmacol. 2013;716(1–3):106 –119.
Guptarak J, Wanchoo S, Durham-Lee J, et al. Inhibition of IL-6
signaling: A novel therapeutic approach to treating spinal cord injury pain. Pain. 2013;154(7):1115–1128.
Won KA, Kim MJ, Yang KY, et al. The Glial-neuronal GRK2 pathway participates in the development of trigeminal neuropathic pain
in rats. J Pain. 2013. doi:10.1016/j.jpain.2013.10.013.
Clark AK, Old EA, Malcangio M. Neuropathic pain and cytokines:
Current perspectives. J Pain Res. 2013;6:803– 814.
Stokes JA, Cheung J, Eddinger K, Corr M, Yaksh TL. Toll-like
receptor signaling adapter proteins govern spread of neuropathic
pain and recovery following nerve injury in male mice. J Neuroinflammation. 2013;10:148.
Ji RR, Berta T, Nedergaard M. Glia and pain: Is chronic pain a
gliopathy? Pain. 2013;154(Suppl 1):S10 –S28.
Zhu X, Cao S, Zhu MD, Liu JQ, Chen JJ, Gao YJ. Contribution of
chemokine CCL2/CCR2 signaling in the dorsal root ganglion and
spinal cord to the maintenance of neuropathic pain in a rat model of
lumbar disc herniation. J Pain. 2014;15(5):516 –526.
endo.endojournals.org
4355
67. Wang D, Couture R, Hong Y. Activated microglia in the spinal cord
underlies diabetic neuropathic pain. Eur J Pharmacol. 2014;728:
59 – 66.
68. Tiwari V, Guan Y, Raja SN. Modulating the delicate glial-neuronal
interactions in neuropathic pain: promises and potential caveats.
Neurosci Biobehav Rev. 2014;45:19 –27.
69. Milligan ED, Watkins LR. Pathological and protective roles of glia
in chronic pain. Nat Rev Neurosci. 2009;10(1):23–26.
70. Arevalo MA, Santos-Galindo M, Acaz-Fonseca E, Azcoitia I, Garcia-Segura LM. Gonadal hormones and the control of reactive gliosis. Horm Behav. 2013;63(2):216 –221.
71. Acaz-Fonseca E, Gonzalez RS, Azcoitia I, Arevalo MA, Garcia-Segura LM. Role of astrocytes in the neuroprotective actions of 17␤estradiol and selective estrogen receptor modulators. Mol Cell Endocrinol. 2014;389(1–2):48 –57.
72. Hatashita S, Sekiguchi M, Kobayashi H, Konno S, Kikuchi S. Contralateral neuropathic pain and neuropathology in dorsal root ganglion and spinal cord following hemilateral nerve injury in rats.
Spine (Phila Pa 1976). 2008;33(12):1344 –1351.