Implication of system xc in neuroinflammation during the onset and maintenance of neuropathic pain

Abstract

Background
Despite the high prevalence of neuropathic pain, treating this neurological disease remains challenging, given the limited efficacy and numerous side effects associated with current therapies. The complexity in patient management is largely attributed to an incomplete understanding of the underlying pathological mechanisms. Central sensitization, that refers to the adaptation of the central nervous system to persistent inflammation and heightened excitatory transmission within pain pathways, stands as a significant contributor to persistent pain. Considering the role of the cystine/glutamate exchanger (also designated as system xc⁻) in modulating glutamate transmission and in supporting neuroinflammatory responses, we investigated the contribution of this exchanger in the development of neuropathic pain.

Methods
We examined the implication of system xc⁻ by evaluating changes in the expression/activity of this exchanger in the dorsal spinal cord of mice after unilateral partial sciatic nerve ligation. In this surgical model of neuropathic pain, we also examined the consequence of the genetic suppression of system xc⁻ (using mice lacking the system xc⁻ specific subunit xCT) or its pharmacological manipulation (using the pharmacological inhibitor sulfasalazine) on the pain-associated behavioral responses. Finally, we assessed the glial activation and the inflammatory response in the spinal cord by measuring mRNA and protein levels of GFAP and selected M1 and M2 microglial markers.

Results
The sciatic nerve lesion was found to upregulate system xc⁻ at the spinal level. The genetic deletion of xCT attenuated both the amplitude and the duration of the pain sensitization after nerve surgery, as evidenced by reduced responses to mechanical and thermal stimuli, and this was accompanied by reduced glial activation. Consistently, pharmacological inhibition of system xc⁻ had an analgesic effect in lesioned mice.

Conclusion
Together, these observations provide evidence for a role of system xc⁻ in the biochemical processes underlying central sensitization. We propose that the reduced hypersensitivity observed in the transgenic mice lacking xCT or in sulfasalazine-treated mice is mediated by a reduced gliosis in the lumbar spinal cord and/or a shift in microglial M1/M2 polarization towards an anti-inflammatory phenotype in the absence of system xc⁻. These findings suggest that drugs targeting system xc⁻ could contribute to prevent or reduce neuropathic pain.

Full text

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Beckers et al. Journal of Neuroinammation (2024) 21:117
https://doi.org/10.1186/s12974-024-03112-9 Journal of Neuroinammation
*Correspondence:
Emmanuel Hermans
emmanuel.hermans@uclouvain.be
Full list of author information is available at the end of the article
Abstract
Background Despite the high prevalence of neuropathic pain, treating this neurological disease remains
challenging, given the limited ecacy and numerous side eects associated with current therapies. The complexity
in patient management is largely attributed to an incomplete understanding of the underlying pathological
mechanisms. Central sensitization, that refers to the adaptation of the central nervous system to persistent
inammation and heightened excitatory transmission within pain pathways, stands as a signicant contributor
to persistent pain. Considering the role of the cystine/glutamate exchanger (also designated as system xc−)
in modulating glutamate transmission and in supporting neuroinammatory responses, we investigated the
contribution of this exchanger in the development of neuropathic pain.
Methods We examined the implication of system xc− by evaluating changes in the expression/activity of this
exchanger in the dorsal spinal cord of mice after unilateral partial sciatic nerve ligation. In this surgical model
of neuropathic pain, we also examined the consequence of the genetic suppression of system xc− (using mice
lacking the system xc− specic subunit xCT) or its pharmacological manipulation (using the pharmacological
inhibitor sulfasalazine) on the pain-associated behavioral responses. Finally, we assessed the glial activation and the
inammatory response in the spinal cord by measuring mRNA and protein levels of GFAP and selected M1 and M2
microglial markers.
Results The sciatic nerve lesion was found to upregulate system xc− at the spinal level. The genetic deletion of
xCT attenuated both the amplitude and the duration of the pain sensitization after nerve surgery, as evidenced
by reduced responses to mechanical and thermal stimuli, and this was accompanied by reduced glial activation.
Consistently, pharmacological inhibition of system xc− had an analgesic eect in lesioned mice.
Conclusion Together, these observations provide evidence for a role of system xc− in the biochemical processes
underlying central sensitization. We propose that the reduced hypersensitivity observed in the transgenic mice
lacking xCT or in sulfasalazine-treated mice is mediated by a reduced gliosis in the lumbar spinal cord and/or a shift in
Implication of system xc−
in neuroinammation during the onset
and maintenance of neuropathic pain
PaulineBeckers1, InêsBelo Do Nascimento1, MathildeCharlier1, NathalieDesmet1, AnnMassie2 and
EmmanuelHermans1*
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Page 2 of 18
Beckers et al. Journal of Neuroinammation (2024) 21:117
Background
Neuropathic pain (NP) largely arises from a pathological
imbalance between excitatory and inhibitory controls of
pain transmission, especially at the level of the dorsal spi-
nal cord [1–4]. Together with a persistent neuroinflam-
mation, this imbalance promotes plastic adaptations in
the central nervous system (CNS) that alter membrane
excitability of neurons. is leads to an enhanced synap-
tic efficacy in pain pathways and is commonly referred
as central sensitization [5]. Pro-inflammatory cytokines
released by glial cells have been suggested to enhance
neuronal and glial release of glutamate, the major excit-
atory neurotransmitter in the CNS [6, 7]. Neuroinflam-
mation also promotes a transcriptional up-regulation of
glutamate receptors, which leads to a persistent facili-
tatory influence on excitatory signals supporting pain
transmission [8].
In different models of NP, the capacity of astrocytes
to rapidly clear glutamate from the synaptic cleft was
shown to be compromised [9–11]. Accordingly, pharma-
cological and genetic interventions aiming at increasing
the expression or the functionality of high-affinity gluta-
mate transporters (EAATs) have demonstrated promis-
ing analgesic effects in animal models of NP [12, 13]. To
date, research on NP and the development of novel treat-
ments targeting glutamate transmission have primarily
focused on receptors and EAATs. As such, other molecu-
lar actors known to play an important role in the control
of glutamate transmission, such as the cystine-glutamate
exchanger (system xc−) have been overlooked.
Mainly expressed in glial cells, system xc− is a trans-
membrane protein composed of two different subunits
[14]. While the heavy chain, 4F2hc (SLC3A2), is shared
among various plasma membrane solute carrier proteins,
the light chain xCT (SLC7A11) determines substrate
specificity and transport activity. System xc− mediates
the exchange of cystine for glutamate in a 1:1 molecular
ratio, releasing glutamate into the extracellular space.
Consequently, it provides cells with the cysteine, the rate-
limiting amino acid residue for synthesizing antioxidant
glutathione, but also indirectly reinforces glutamate sig-
naling [15, 16]. Hence, system xc− has even been identi-
fied as the major source of extracellular glutamate in
several structures of the CNS [17, 18]. As for EAATs, a
tight regulation of its expression and activity is essential
to support protection against oxidative stress, but also to
control extracellular glutamate concentration and neuro-
nal excitation.
Considering its biochemical functions and its wide-
spread distribution in the CNS, system xc− has been
studied in several neurological disorders in which dys-
regulated excitatory transmission contributes to nervous
insults and neurodegeneration. In particular, increased
xCT expression has been documented in models of Par-
kinson’s disease, amyotrophic lateral sclerosis, multiple
sclerosis as well as in stroke or glioblastoma [19–24]. A
role for system xc− was also documented in the modula-
tion of inflammatory responses, raising further interest
for its implication in pathologies combining neuroin-
flammation and increased neuronal excitation [19]. Con-
sidering this, the present study aimed at examining a
putative role for system xc− in the modulation of noci-
ceptive signals and associated neuroinflammation. To
address this question, we have used a validated surgical
mouse model of NP in which the expression and activity
of system xc− were manipulated by genetic and pharma-
cological approaches.
Materials and methods
Animals and ethical statement
All experiments were conducted in strict accordance
with the recommendations of the European commis-
sion and with the agreement of the Belgian Ministry
of Agriculture (code number LA 1,230,618). e ethi-
cal committee of the Université catholique de Louvain
for animal experiments specifically approved this study
(aggregation number 2019/UCL/MD/033). Wild-type
(xCT+/+) and xCT knock-out (xCT−/−) mice used in this
study were high-generation descendants (with C57BL/6J
background) of the strain previously described [25].
ese mice lacking xCT were generously provided by Pr.
Hideyo Sato (Department of Medical Technology, Niigata
University, Japan) and bred in the animal facilities of the
Vrije Universiteit Brussel and the Université catholique
de Louvain. All experiments combined homozygous ani-
mals obtained both from the breeding of heterozygous
animals (F1) and from a second generation using homo-
zygous parents (F2).
All animals were accommodated under standard labo-
ratory conditions, receiving food and water ad libitum,
and were housed in the animal facility at the Université
catholique de Louvain, in a 12–12 h light-dark cycle,
controlled temperature and humidity conditions. xCT
knock-out mice were genotyped by end-point PCR on
genomic DNA extracted from an ear biopsy using spe-
cific primers for the Slc7a11 gene. PCR amplification
products were analyzed by agarose-gel electrophoresis
microglial M1/M2 polarization towards an anti-inammatory phenotype in the absence of system xc−. These ndings
suggest that drugs targeting system xc− could contribute to prevent or reduce neuropathic pain.
Keywords xCT, Slc7a11, Spinal cord, Central sensitization, Glutamate, Inammation, Glial cells, Chronic pain
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Page 3 of 18
Beckers et al. Journal of Neuroinammation (2024) 21:117
to identify bands revealing the intact or truncated gene
[25]. Since pain hypersensitivity in model of NP is sex-
dependent, only female mice were herein used to reduce
variability.
Neuropathic pain model – partial sciatic nerve ligation
(PSNL)
Adult female mice aged between 10 and 12 weeks were
randomly subjected to PSNL or sham surgery using a
procedure described earlier with minor modifications
[26]. Briefly, mice were anesthetized with sevoflurane (4%
for induction and 3% for maintenance with oxygen as a
carrier gas). After isolation from the surrounding con-
nective tissue, the left sciatic nerve was exposed. With an
8 − 0 suture, one half to one third of the nerve was tightly
ligated above its trifurcation. Muscle and skin layers were
finally closed with a 6 − 0 suture. Sham surgeries were
performed by simply exposing the nerve before suturing
the wound without performing the sciatic nerve ligation.
Mechanical hypersensitivity – von frey hair lament test
Animals were placed in transparent chambers positioned
on an elevated mesh floor. Acclimatization was allowed
for 20min after which the von Frey test was performed.
Herein, a set of 10 calibrated von Frey hair filaments
(Stoelting Co., Wood Dale, IL) was used (0.04, 0.07, 0.16,
0.4, 0.6, 1, 1.4, 2, 4 and 6g). Filaments were applied per-
pendicular to the plantar surface of the hind paw starting
with the 0.4g filament.
To determine the mechanical sensitivity, the “up and
down” method was used [27]. Briefly, the choice for the
following filament was based on the previous filament
response, being the closest-lower filament in case of a
positive response or the closest-higher filament in case
of a negative response. A positive response was defined
by a paw withdrawal associated with aversive behavior,
such as licking and/or shaking the stimulated paw. is
method of von Frey filament application was continued
until a sequence of nine filament applications was com-
pleted. e 50% paw withdrawal threshold (PWT) was
then calculated using the formula previously described
[28]. e experimenter responsible for conducting the
behavioral tests was blind to the treatment, the genotype
and the surgery performed on the mice. e cumulative
changes in PWT from baseline values throughout the
entire experiments have been computed and are depicted
as the area under the curve (AUC).
Thermal hypersensitivity – hargreaves Test
e thermal paw withdrawal latency (PWL) was used
to assess hyperalgesia [29]. Briefly mice were acclimated
for 20min in separated transparent, bottom-free plastic
chambers placed over the glass enclosure of the Harg-
reaves paw thermal stimulator (University of California,
San Diego, CA). A heat source was positioned under-
neath the plantar surface of the hind paw (both ipsi- and
contralaterally) and the time taken to withdraw from the
heat source was automatically recorded by the apparatus.
Each animal was stimulated 3 times with a 3min inter-
stimulation period. e PWL is averaged per animal
and per paw. A cut-off was fixed at 20s of stimulation to
avoid any tissue damage. e experimenter responsible
for conducting the behavioral tests was blind to the treat-
ment, the genotype and the surgery performed on the
mice. As mentioned before, PWL data have been com-
puted as the AUC.
Sulfasalazine administration
Animals were randomly assigned to either the treat-
ment or control group. An 80mg/mL stock solution of
sulfasalazine (SAS, Sigma-Aldrich, St. Louis, MO) was
prepared by dissolving the powder in DMSO. e final
solution was daily prepared by adding saline to reach the
desired concentration of SAS. e pH was adjusted using
a small volume of NaOH 0.1M. Mice were daily injected
intraperitoneally (i. p.) with 200mg/kg of SAS (or vehicle
that includes the same dilution of DMSO for the con-
trols) [30, 31] starting 2 days prior the sciatic nerve liga-
tion and during the entire experiment until sacrifice, 3 or
7 days after surgery.
Tissue collection
Mice were euthanized 3 or 7 days after sciatic nerve or
sham surgery with CO2 and the spinal cord was imme-
diately extruded with phosphate-buffered saline (PBS)
as previously described [32]. e tissue from the lumbar
enlargement of the spinal cord was isolated. A first longi-
tudinal cut was performed along the ventral-median fis-
sure exposing two halves corresponding to the ipsi- and
contralateral sides. Another longitudinal cut along the
central canal of each halves was performed to collect four
quadrants. e dorsal quadrants were rapidly frozen in
liquid nitrogen and finally stored at -80°C before being
processed for RNA extraction or synaptosome prepara-
tion. For immunohistochemistry, animals were trans-
cardialy perfused with PBS before lumbar spinal cord
collection.
Total RNA extraction and real-time quantitative PCR
(RT-qPCR)
Ipsi- and contralateral lumbar dorsal spinal cord sam-
ples were frozen and mechanically dissociated using a
pre-chilled Teflon/glass homogenizer in TriPure iso-
lation reagent (Roche, Bâle, Switzerland), followed by
RNA extraction according to the manufacturer’s proto-
col. Reverse transcription was carried out with 1 µg of
extracted RNA using the iScript cDNA synthesis kit (Bio-
Rad Laboratories, Hercules, CA) in a total volume of 20
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Page 4 of 18
Beckers et al. Journal of Neuroinammation (2024) 21:117
µL. Real-time qPCR amplifications were then performed
using a 3-steps protocol with the Bio-Rad CFX Connect™
real-time PCR detection system (Bio-Rad Laboratories),
in a total volume of 20 µL containing iTaq Universal
SYBR® Green Supermix (Bio-Rad Laboratories), cDNA
(equivalent to 10 ng retrotranscribed RNA) and a final
concentration of 0.5 µM of each primer. Quantitative
analysis was performed using the delta-delta Ct method,
normalized to the relative expression of 3 different
housekeeping genes (Ywhaz, RPL-19 and HPRT-1) previ-
ously validated for RT-qPCR studies on injured nervous
tissues [33, 34]. e sequences of the primers used in this
study are the following: for xCT, forward 5′- G A T G C T G
T G C T T G G T C T T G A − 3′ and reverse 5′- G C C T A C C A T
G A G C A G C T T T C − 3′; for GFAP, forward 5’- G A G G G A
C A A C T T T G C A C A G G − 3’ and reverse 5’- T C C T C C A G
C G A T T C A A C C T T − 3’; for Iba-1, forward 5’- G G A T T T
G C A G G G A G G A A A A G − 3’ and reverse 5’- T G G G A T C
A T C G A G G A A T T G − 3’; for NOX-2, forward 5’- T G A A
T G C C A G A G T C G G G A T T T − 3’ and reverse 5’- C C C C C
T T C A G G G T T C T T G A T T T − 3’; for Arg-1, forward 5’- C
G T G T A C A T T G G C T T G C G A G − 3’ and reverse 5’- A T C
A C C T T G C C A A T C C C C A G − 3’; for RPL-19, forward 5’-
T G A C C T G G A T G A G A A G G A T G A G − 3’ and reverse 5’-
C T G T G A T A C A T A T G G C G G T C A A T C − 3’; for HPRT-1,
forward 5’- C C T A A G A T G A G C G C A A G T T G A A − 3’ and
reverse 5’- C C A C A G G A C T A G A A C A C C T G C T A A − 3’;
for Ywhaz, forward 5’- T A G G T C A T C G T G G A G G G T C G
− 3’ and reverse 5’- G A A G C A T T G G G G A T C A A G A A C T
T − 3 ’.
Crude synaptosome preparation and [3H]-
l
-glutamate
uptake assay
Frozen dorsal spinal cord samples were used for synap-
tosome preparation and for the measurement of system
xc− driven [3H]--glutamate transport. e method is
based on the reversal uptake of glutamate when per-
formed in the absence of extracellular cystine and in the
absence of Na+, as validated in our previous study [35].
Briefly, the tissue was homogenized and centrifuged in
ice-cold isotonic solution. e final pellet containing the
synaptosomes was diluted in ice-cold buffer lacking Na+,
in order to avoid any detection of EAAT-mediated gluta-
mate uptake. Protein concentration was determined, and
samples were diluted to achieve a final protein concentra-
tion of 33.33µg/mL.
To assess the uptake, [3H]--glutamate (with spe-
cific activity of 48.6 Ci/mmol, Perkin Elmer, Waltham,
MA) was used as substrate at a concentration of 20 nM.
In a total volume of 500 µL, 10µg of the synaptosome
preparation was incubated with the substrate at 37 °C
in a 96-well Masterblock (Greiner Bio-one, Kremsmün-
ster, Austria). When indicated homocysteic acid (HCA,
H9633, Sigma-Aldrich), a system xc− inhibitor, was added
to evaluate the HCA-dependent uptake. After 20 min
incubation at 37°C, the suspension was filtered (UniFilter
GF/B, PerkinElmer), and washed with ice-cold Na+-free
buffer. After drying overnight, a liquid scintillation solu-
tion, Microscint 20 (PerkinElmer) was added to each well
and the radioactivity measurements were done using the
Topcount® NXT Microplate scintillation and lumines-
cence counter (PerkinElmer). Results are expressed as a
percentage of the respective [3H]--glutamate uptake in
the contralateral side.
Immunohistochemistry
Lumbar spinal cords were used for immunohistochem-
istry as previously described by Gallo et al. with minor
modifications [36]. Briefly, after tissue collection, lum-
bar spinal cords were fixed overnight in 4% parafor-
maldehyde in PBS. Cryoconservation was achieved by
successively incubating the samples in 10%, 20% and
30% sucrose/PBS solution. Afterwards, spinal cords were
frozen and stored at -80°C until further use. For cryo-
sectioning, samples were embedded in tissue Tek O.C.T
(Sakura, Osaka, Japan) and transversal sections of 20m
were cut using a CM3050S Leica cryostat (Leica Micro-
systems, Wetzlar, Germany). Cryosections were collected
on Superfrost Plus object glass slides (ermo Fisher Sci-
entific, Waltham, MA) and stored at -20°C until immu-
nohistological staining. After a dry time of 1h at room
temperature (RT), sections were washed in PBS and incu-
bated in a blocking buffer (5% normal goat serum, 0.5%
Triton in PBS) for 1h at RT. Sections were then incu-
bated overnight at 4°C with either of the following pri-
mary antibodies diluted in the blocking buffer: anti-Iba1
(rabbit, 1:1.000, Wako, Osaka, Japan, cat. no. 019/19,741
provided at 0.5mg/mL) or anti-GFAP (chicken, 1:1.000,
Abcam, Cambridge, UK, cat. no. ab4674 provided at
1 mg/mL). e next day, sections were rinsed in PBS,
and were incubated for 2 h at RT with anti-rabbit or
anti-chicken secondary antibodies conjugated to Alexa
Fluor 647 (ermo Fisher Scientific A21245 and A21449,
respectively, both provided at 2mg/ml and diluted 1:500
in PBS). Afterwards, sections were again washed with
PBS and mounted on glass slides using EverBrite mount-
ing medium (Biotium, Fremont, CA). Images were exam-
ined with an EVOS fluorescence microscope (AMG
EVOS fl., Westburg, Leusden, Netherlands). e fluores-
cent intensity was quantified using the mean grey value
of the defined area (ImageJ software).
Statistical analyses
Data from biochemical studies (uptake assay, RT-qPCR,
immunohistochemistry, and AUC), statistical analyses
were performed on the difference in log-transformed
ipsilateral versus contralateral measures for each mouse
in order to control for individual variability. Normality
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Page 5 of 18
Beckers et al. Journal of Neuroinammation (2024) 21:117
was assessed using the Shapiro-Wilk normality test, and
equality of variances was tested using the Levene test.
Graphs depicting the back-transformed data are pro-
vided for all experiments to facilitate interpretation. As
such, results are presented as geometric mean for bio-
chemical endpoints and as arithmetic mean for behav-
ioral endpoints. Statistical analyses were performed using
one or two-way ANOVA model, depending on the exper-
imental design. In case where a significant interaction
was observed in the two-way ANOVA model, Tukey’s
pairwise comparison method was employed. When
appropriate and identified by the Grubb’s test, significant
outliers were removed from the analysis set. For all analy-
ses, a nominal two-sided Type I error of 0.05 was consid-
ered for statistical significance. Statistical analyses were
conducted using GraphPad Prism 9.5.1 and JMP Pro 17
softwares.
Results
PSNL triggers increased xCT expression and system xc-
activity in the ipsilateral spinal cord
To examine the contribution of system xc− in NP, the
impact of a peripheral nerve lesion on the expression
of the specific subunit xCT and system xc− activity was
assessed by RT-qPCR and 3H-glutamate uptake, respec-
tively. Animals were sacrificed 3 days or 7 days after
surgery and quadrant at the ipsi- and contralateral lum-
bar dorsal spinal cord were dissected and processed for
analyses. ree days after PSNL, xCT mRNA level was
significantly increased in the ipsilateral dorsal quadrant
of the lumbar spinal cord. As shown in Fig.1A and B, a
70% and 20% increase in lesioned mice as compared to
the sham-operated mice was measured for xCT expres-
sion and system xc− activity, respectively. is change in
xCT expression appeared transient as it was absent from
samples collected 7 days after surgery (Fig.1C). However,
the increased system xc− activity was even more impor-
tant in synaptosomes prepared from spinal cord samples
7 days after surgery (130% increase in the ipsilateral dor-
sal quadrant of lesioned mice as compared to the sham-
operated mice) (Fig.1D).
Mice lacking xCT present a higher pain tolerance following
PSNL
e role of system xc− in the development of pain sen-
sitivity was examined by subjecting wild-type (xCT+/+)
and xCT-deficient (xCT−/−) mice to unilateral PSNL.
Female mice were monitored for 7 days post-injury using
a von Frey (Fig.2A-C) and a Hargreaves test (Fig. 2D-
F) to respectively evaluate lesion-associated allodynia
and hyperalgesia. In the absence of nerve lesion, mice
lacking xCT did not present any signs of hypersensitiv-
ity to mechanical or thermal stimuli compared to their
wild-type littermates. Also, sham-operated xCT−/− mice
showed similar PWT (Fig. 2A-C) and PWL (Fig.2D-F)
as compared to non-lesioned xCT+/+ mice. As shown in
Fig. 2A, nerve-lesioned xCT+/+ animals exhibited pro-
longed and strong mechanical allodynia starting from
day 1 after surgery. e 50% PWT reached the minimum
value of 0.19 ± 0.04g in injured xCT+/+ animals compared
to 3.49 ± 0.26 g in the sham-operated animals. In mice
lacking xCT, we also observed a decreased threshold fol-
lowing the sciatic nerve ligation (1.39 ± 0.21 g compared
to 3.22 ± 0.32 g for sham-operated animals). However,
this decrease was less pronounced compared to what
was observed in xCT+/+ operated animals (Fig.2A&B).
Moreover, the PWT to mechanical stimulation returned
to baseline values as from the sixth day after surgery,
indicating that the hypersensitivity observed in xCT−/−
animals was transient. At variance, the decreased pain
threshold observed in the xCT+/+ injured mice persisted
for the entire duration of the experiment. No changes
were observed in the contralateral paw when tested for
mechanical sensitivity (Fig.2C).
When testing the response to a thermal stimulus
using the Hargreaves test, xCT+/+ mice undergoing
PSNL developed a strong and persistent hyperalgesia
as indicated by the decreased latency for ipsilateral paw
withdrawal reaching 1.63 ± 0.11 s at 2 days post-injury
(Fig.2D). In contrast, mice lacking xCT did not develop
any thermal hyperalgesia. Indeed, at all tested time points
following the surgery, the PWL of injured xCT−/− mice
was similar to what was observed in sham-operated ani-
mals (AUC of 19.98 ± 0.39 for xCT−/− injured mice and
21.75 ± 0.75 for uninjured xCT−/− mice) (Fig. 2E). No
changes were observed when testing the contralateral
hind paw for thermal sensitivity (Fig.2F).
xCT-/- mice show reduced astrogliosis and a distinct
microglial polarization after PSNL
Pain sensitization following peripheral nerve injury often
coincides with pronounced glial activation in the ipsi-
lateral spinal cord. Astroglial and microglial activations
are commonly authenticated by evidencing an increased
expression of the typical markers glial fibrillary acidic
protein (GFAP) and ionized calcium-binding adapter
molecule 1 (Iba-1), respectively. Changes in the protein
expression (Fig.3) and mRNA (Fig.4) levels of both glial
markers were therefore examined by immunohistochem-
istry on lumbar spinal cord sections and RT-qPCR on
the dorsal quadrants of the lumbar spinal cord of xCT+/+
and xCT−/− mice undergoing PSNL or sham surgeries.
As expected, GFAP was significantly upregulated (1.5
fold-increase for protein level and up to 3 fold-increase
for mRNA) in the spinal cord of xCT+/+ mice 3 days after
unilateral surgery as compared to the contralateral side
or to sham-operated animals (Fig.3A, amp and B A). is
was however not observed in samples collected 7 days
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Page 6 of 18
Beckers et al. Journal of Neuroinammation (2024) 21:117
after the surgery (Figs.3C and 4B). e early upregula-
tion of GFAP was less pronounced (1.6 fold-increase for
mRNA) in xCT−/− mice indicating a lower astrocytic
activation following nerve surgery (Fig.3B A).
Regarding microglial activation, a 2-fold increase in
Iba-1 mRNA level was observed in the ipsilateral spinal
cord of both xCT+/+ and xCT−/− mice, 3- and 7-days
post-surgery compared to the contralateral side or to the
sham-operated animals (Fig. 4C&D). Iba-1 immunore-
activity was found to be increased (1.2-fold after 3 days
and 1.6-fold after 7 days) in the ipsilateral spinal cord
of xCT+/+ animals following PSNL compared to sham-
operated animals or to the contralateral side. is, how-
ever, was not observed in transgenic animals lacking xCT
where no alteration was detected (Fig.3D&E).
Upon gliosis, microglial cells change their expression
of several inflammation-related genes, and thereby adopt
predominantly pro- or anti-inflammatory phenotypes
[37]. e expression of NADPH oxidase 2 (NOX-2) and
arginase 1 (Arg-1) was examined to monitor the polariza-
tion of reactive microglial cells in the spinal cord of mice
after PSNL (Fig.4E-H). In the ipsilateral dorsal quadrant
Fig. 1 xCT expression and activity in the lumbar dorsal spinal cord following PSNL or sham surgery. Three (panel A & B) and seven (panel C & D) days
post-surgery, the ipsilateral and contralateral dorsal horn quadrants of the lumbar spinal cord were dissected and used for RT-qPCR or functional assay.
xCT mRNA was quantied, normalized to the mean of 3 housekeeping genes (RPL-19, Ywhaz, HPRT-1) and expressed as a percentage of the respective
contralateral mRNA level (A, C). Uptake of 3H-L-glutamate on crude spinal synaptosomes was used to determine system xc− functionality (B, D). Ipsilateral
system xc− specic uptake (HCA-dependent) is expressed as relative (%) to the respective contralateral side. As shown on the gure, data were obtained
from 5 to 7 dierent animals per group and are presented as geometric mean with SD. Statistical analyses were conducted using a one-way ANOVA
model. PSNL partial sciatic nerve ligation; Glu glutamate; HCA homocysteic acid
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Beckers et al. Journal of Neuroinammation (2024) 21:117
Fig. 2 Allodynia and hyperalgesia in xCT+/+ and xC T−/− mice following PSNL or sham surgery. Pain hypersensitivity was assessed upon stimulation of the
ipsilateral (A, B, D, E) and contralateral (C, F) hind paw at baseline (BL) and for up to 7 days after surgery. The 50% PWT to mechanical stimulation was
used to determine allodynia (A-C), whereas the PWL to thermal stimulation was used as a read-out of hyperalgesia (D-F). Panel B and E depict the area
under the curve (AUC in arbitrary unit a.u.) of the ipsilateral 50% PWT and PWL, respectively. Data are presented as arithmetic mean with SEM (A, C, D,
F) or geometric mean with SD (B, E) of 5 animals per group for allodynia and 7 animals per group for hyperalgesia. Statistical analyses were performed
through a two-way ANOVA model (B, E) with repeated measures (A, C, D, F). Main and interaction eects are indicated below the graphs. When statisti-
cally signicant interaction was detected, Tukey’s pairwise comparisons were conducted. P-values for signicant eects are reported on the graphs (B, E).
In panel A and D, * indicates a signicant dierence between lesioned wild-type (PSNL xCT+/+) and transgenic mice lacking xCT (PSNL xCT−/−), # indicate
a signicant dierence between lesioned and non-lesioned animals (within the same genotype). PSNL partial sciatic nerve ligation; PWT paw withdrawal
threshold; PWL paw withdrawal latency; AUC area under the curve; S surgery eect; G genotype eect; S*G interaction eect
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Fig. 3 (See legend on next page.)
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Page 9 of 18
Beckers et al. Journal of Neuroinammation (2024) 21:117
of the lumbar spinal cord of xCT+/+ mice, 3 days after
surgery, the expression of the pro-inflammatory marker
NOX-2 was upregulated (2.8-fold increase) while the
anti-inflammatory gene Arg-1 was downregulated (2-fold
decrease). At variance, a trend for an upregulation of
NOX-2 (1.5-fold increase) was detected in the ipsilateral
spinal cord of xCT−/− mice after PSNL, while no changes
were observed regarding Arg-1 mRNA level (Fig.4E&G).
Additionally, in the lumbar spinal cord of xCT+/+ and
xCT−/− mice the expression of both inflammation-related
microglial markers were unchanged when examined 7
days post-surgery (Fig.4F&H).
Sulfasalazine has an anti-allodynic and anti-hyperalgesic
eect
To assess the potential of targeting system xc− in prevent-
ing the development of NP, a pharmacological approach
was employed. Both xCT+/+ and xCT−/− mice subjected
to PSNL surgery were treated daily with the inhibitor of
system xc−, SAS, or with vehicle. Treatment was initi-
ated 2 days before PSNL and pursued until sacrifice. Mice
were monitored daily for allodynia and hyperalgesia for
up to 3 or 7 days after surgery, at which stage spinal cord
tissue was collected for RT-qPCR analysis. As mentioned
before, 3 days after PSNL, the mRNA level of xCT was
significantly increased in the ipsilateral dorsal quadrant
of the lumbar spinal cord (2-fold increase as compared to
the contralateral quadrant). is change in xCT expres-
sion was nearly completely reversed by the administra-
tion of SAS (Fig.5A). Also, this effect appeared transient
as xCT mRNA level remained unchanged 7 days post-
surgery and SAS treatment was without influence at that
stage (Fig.5B).
Regarding the influence of the treatment on the pain-
associated behaviors, it was observed that baseline mea-
surements of mechanical and thermal pain thresholds
remained unaltered by SAS treatment in the absence of
surgery (Fig.5C-H). Consistent with here-above detailed
data, vehicle-treated xCT+/+ mice undergoing PSNL pre-
sented a robust ipsilateral mechanical allodynia (Fig.5C-
E) and thermal hyperalgesia (Fig. 5F-G) that persisted
for at least 7 days. Conversely, xCT+/+ mice receiving
SAS showed a reduced hypersensitivity to the mechani-
cal stimulus as they presented a higher 50% PWT com-
pared to vehicle-treated mice (AUC of 11.93 ± 0.23 for
SAS-treated mice and 8.18 ± 0.53 for control group,
p < 0.0001) (Fig.5C&D). No changes were observed when
testing the contralateral paw for mechanical sensitivity
(Fig.5E). Besides, when challenged with thermal stimula-
tion, SAS-treated xCT+/+ mice did not develop any signs
of thermal hypersensitivity. Indeed, at all tested time
points, the PWL was similar to the values observed at
baseline (Fig.5F-H). To confirm that the observed effects
were specifically driven by the inhibition of system xc−,
lesioned-mice lacking xCT were also subjected to the
same SAS treatment. In both behavioral tests, no sig-
nificant differences in the pain thresholds were observed
when comparing SAS- and vehicle-treated xCT−/− mice.
Sulfasalazine reduces astrocytic and microglial activation
after PSNL
e effects of SAS treatment on the PSNL-induced glial
activation were examined by immunohistochemistry
(Fig.6) and RT-qPCR (Fig.7) for GFAP and Iba-1, in the
dorsal quadrants of the lumbar spinal cord of xCT+/+
mice daily treated with either SAS or vehicle. As pre-
viously described, lesioned-mice displayed increased
ipsilateral expression of GFAP and Iba-1 (Fig.6A). Intra-
peritoneal treatment with SAS strongly reduced immu-
noreactivity of both glial markers (Fig.6B-E) particularly
with respect to Iba-1 staining, 7 days after the surgery
(Fig.6E). ese changes were not observed by RT-qPCR
(Fig.7A-D). However, pharmacological inhibition of sys-
tem xc− leads to a significant reduction of NOX-2 expres-
sion (from 333.19 ± 51.54% for the vehicle-treated mice
to 109.86 ± 40.76% for the SAS-treated group 3 days after
the surgery, p < 0.01) (Fig. 7E&F). No significant differ-
ence was observed concerning the expression of Arg-1,
the marker used to monitor the anti-inflammatory phe-
notype of microglia (Fig.7G&H).
Discussion
Peripheral nerve injuries often trigger maladaptive plas-
ticity in nociceptive pathways, resulting in the patho-
logical amplification of excitatory transmission. is is
well documented in the dorsal horn of the spinal cord,
which serves as the first relay center for integrating and
regulating nociceptive information, contributing to the
development of chronic pain [5, 38]. e reinforcement
of glutamate transmission can be attributed to different
mechanisms, such as an enhanced neuronal release of
glutamate causing an increased activation of glutamate
receptors and associated downstream signaling path-
ways [39, 40]. Besides, impaired glutamate clearance by
(See gure on previous page.)
Fig. 3 Glial activation in the spinal cord following PSNL or sham surgery in xCT+/+ and xCT−/− mice. Immunohistological stainings for GFAP and Iba-1
were analyzed 3 (A, B, D) and 7 (A, C, E) days after the surgery in samples from wild-type or transgenic mice lacking xCT (A). The uorescent intensity for
GFAP (B, C) and Iba-1 (D, E) in the dorsal horns was quantied. The results from the ipsilateral side are normalized and expressed as a percentage of the
respective contralateral side. Images shown are representative of 5 dierent animals and histograms represent the geometric means with SD. Statistical
analyses were performed through a two-way ANOVA model. Main and interaction eects are indicated below the graphs. When statistically signicant
interaction was detected, Tukey’s pairwise comparisons were conducted. P-values for signicant eects are reported on the graphs. PSNL partial sciatic
nerve ligation; GFAP glial brillary acidic protein; Iba-1 ionized calcium binding adapter protein 1; S surgery eect; G genotype eect; S*G interaction eect
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Beckers et al. Journal of Neuroinammation (2024) 21:117
Fig. 4 (See legend on next page.)
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Beckers et al. Journal of Neuroinammation (2024) 21:117
high affinity glial transporters is also documented [9,
10]. Modulating the molecular mechanisms that regu-
late glutamate transmission holds promise in preventing
or alleviating chronic pain. Indeed, drugs that enhance
glutamate transporter activity or antagonizing gluta-
mate receptors were proven effective in models of NP,
but potential side effects would restrict their clinical use
[41–43].
In addition to extensive research on glutamate trans-
porters, accumulating evidence has recently emphasized
the role of system xc− in modulating glutamate transmis-
sion [44]. Widely distributed throughout the CNS, this
cystine-glutamate exchanger responsible for the non-
vesicular glutamate release is not only implicated in the
modulation of excitatory signals, but also in the defense
against oxidative stress and neuroinflammation [45, 46].
is dual role played by system xc− supports a putative
involvement in diverse neurological pathologies, where
both dysregulation of glutamate signaling and persis-
tent neuroinflammation contribute to neuronal damages
[15]. is was also evidenced in a model of bone cancer
pain where increased expression of system xc− supports
the release of glutamate, contributing to the activation of
sensory neurons [47]. In this context, the present study
using a model of peripheral nerve lesion provides com-
pelling evidence on the role played by this exchanger in
the physio-pathological mechanisms of NP.
In both rats and mice, the PSNL is a commonly used
model of surgical nerve lesion to study NP. Within a few
hours after the nerve ligation, rodents exhibit signs of
thermal hyperalgesia, mechanical allodynia and spon-
taneous pain that persist for several months [48]. is
lesion has been reported to trigger plasticity in the pri-
mary afferent fibers and sustained neuroinflammation in
the dorsal lumbar spinal cord, a hallmark of central sen-
sitization [10]. In the present study, the invalidation of
system xc− appeared to influence both the amplitude and
the duration of the pain sensitization after PSNL. Com-
pared to wild-type mice, xCT knock-out mice exhibited
reduced sensitivity to mechanical stimulation from the
first day post-surgery, with pain sensitivity returning to
baseline within just 6 days.
While the nociceptive behavior of mice lacking xCT
was improved at both early and late stages after ligation,
it is noteworthy that the upregulation of xCT mRNA
in the spinal cord of wild-type animals was primarily
observed at early stage after PSNL. is suggests a pre-
dominant implication of system xc− at the onset of the
sensitization process. Hence, in the model of bone can-
cer-induced pain, the inhibition of system xc− was shown
to delay the onset of nociception [47]. Accumulating evi-
dence indicates that an inadequate management of acute
pain can lead to its chronicisation and that early changes
in the pain pathways can lead to later plastic changes
responsible for persistent pain [49]. is putative impli-
cation of xCT at the onset of NP is consistent with the
observation that knock-out mice do not develop any
sign of thermal hyperalgesia following PSNL. Indeed, if
xCT+/+ and xCT−/− mice had initially exhibited similar
hypersensitivity within a few hours but showed a faster
resolution in mice lacking xCT, it would suggest a later
involvement of system xc−. While further experiments
are needed to ascertain the specific role of system xc− in
the different stages of NP. Our observations hold signifi-
cant implication regarding the opportunity to target sys-
tem xc− in the management or prevention of NP.
e beneficial effect of the genetic suppression of
xCT on the development and persistence of allodynia
and hyperalgesia in the surgical model of NP is consis-
tent with the observation that such lesion triggers an
increased expression of xCT in the dorsal spinal cord.
Nevertheless, the use of a constitutive transgenic mice
strain in which xCT expression is absent during the
entire animal development raises questions related to
compensatory mechanisms [50]. To validate the findings
from xCT-deficient animals, the pharmacological inhi-
bition of system xc− in adult mice constitutes a relevant
alternative approach. Indeed, considering the upregu-
lation of xCT and the enhanced system xc− activity fol-
lowing sciatic nerve ligation, targeting this exchanger
might help to reduce NP symptoms. Nowadays, the
most used inhibitor of system xc− is SAS, an EMA- and
FDA-approved drug prescribed to treat inflammatory
diseases of the intestine [51, 52]. Effectiveness in these
disorders arises from its breakdown by the gut micro-
biota, generating anti-inflammatory metabolites. As only
the intact parent molecule was proven effective in inhib-
iting system xc− [52–54], SAS was herein administrated
(See gure on previous page.)
Fig. 4 Assessment of spinal mRNA of glial markers following PSNL or sham surgery in xC T+/+ and xCT−/− mice. Three (left panels) and seven (right panels)
days post-surgery, the ipsilateral and contralateral quadrants of the dorsal horn of the lumbar spinal cord were dissected and used for RT-qPCR analyses.
Specic markers were used to evaluate astrocytic (A, B) and microglial (C, D) activation, GFAP and Iba-1 respectively. Other markers were used to assess
microglia activation prole towards pro-inammatory (NOX-2: panel E & F) and anti-inammatory (Arg-1: panel G & H) phenotype. For all markers, the
mRNA was quantied, normalized to the mean of 3 housekeeping genes (RPL-19, Ywhaz, HPRT-1) and expressed as a percentage of the respective con-
tralateral mRNA level. As shown on the gure, data were obtained from 5 to 7 dierent animals per group and are presented as geometric mean with SD.
Statistical analyses were performed through a two-way ANOVA model. Main and interaction eects are indicated below the graphs. When statistically
signicant interaction was detected, Tukey’s pairwise comparisons were conducted. P-values for signicant eects are reported on the graphs. PSNL
partial sciatic nerve ligation; GFAP glial brillary acidic protein; Iba-1 ionized calcium binding adapter protein 1; NOX-2 NADPH oxidase 2; Arg-1 Arginase 1;
S surgery eect; G genotype eect; S*G interaction eect
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Beckers et al. Journal of Neuroinammation (2024) 21:117
Fig. 5 (See legend on next page.)
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Page 13 of 18
Beckers et al. Journal of Neuroinammation (2024) 21:117
intraperitoneally to reduce its metabolization. Even
though some papers reported on a limited capacity of
SAS to cross the blood-brain barrier, several researches
have demonstrated beneficial effects in nervous disor-
ders affecting both the brain and the spinal cord such as
reduced microglial activation and reduced tumor growth
in mouse model of glioblastoma [53, 55, 56]. Given that
system xc− putatively participates in the onset of NP, SAS
was administered preventively, starting 2 days before the
lesion. In these conditions, a robust anti-allodynic and
anti-hyperalgesic effect of SAS was observed in xCT+/+
mice undergoing PSNL. Even though these data are con-
sistent with the results obtained with knock-out animals,
pointing out xCT as a key actor in the pain sensitization,
a possible anti-inflammatory component of the treatment
cannot be ruled out. us, some evidence indicated that
SAS itself possess anti-inflammatory properties through
an inhibition of the transcription factor NF-κB involved
in immune responses [57, 58]. Surprisingly, SAS, being
a system xc− inhibitor, led to a downregulation of xCT
mRNA. is post-translational regulation might be
attributed to its anti-inflammatory properties, resulting
in reduced recruitment of astrocytes and microglia which
are known to upregulate xCT during inflammatory con-
ditions. To confirm the specific role of xCT, SAS was
administered to xCT−/− mice, but no analgesic effect was
observed compared to the vehicle-treated xCT−/− mice.
Together, these data underscore the relevance of tar-
geting system xc− in NP but call for the development of
more specific inhibitors to mitigate potential side effects
as previously reported with SAS [30].
Knowing that system xc− is mainly expressed in glial
cells and that over the past decades, both peripheral and
central glia and immune cells have taken center stage in
research on NP, the specific implication of astrocytes and
microglial cells was examined. e upregulation of xCT
following PSNL surgery, was correlated with the acti-
vation of astrocytes and microglia in the dorsal spinal
cord. Resident glial cells actively participate in the strong
inflammatory reaction in models of NP by releasing
mediators such as TNFα, interleukins, chemokines, ATP.
is neuroinflammatory cascade is further propagated
by the recruitment of microglial cells and astrocytes [59].
After both peripheral or central nerve lesions, these cells
promote neuroinflammation at several levels of the pain
pathway leading to the onset and progression of pain
hypersensitivity. While Iba-1 is primarily recognized for
its role in the initiation of NP and GFAP for its mainte-
nance [3, 60], results are not always straightforward as
temporal dynamic changes may differ across pain models
or spinal regions [61]. At variance with several published
data, our findings unveil a rapid and transient upregula-
tion of GFAP early after the nerve lesion, prompting fur-
ther investigation into the underlying chronology of this
glial response. In line with the existing literature, our
experimental findings regarding the persistent upregu-
lation of Iba-1 imply that microglial activation may per-
sist for an extended period, potentially even beyond pain
resolution [62]. Emerging evidence suggests that specific
subtypes of immune cells can reduce pain and contrib-
ute to the resolution of NP [48, 63]. Previous studies
have evidenced that activated microglia show increased
system xc− expression and that the genetic invalidation
of xCT alters their polarization profile, predominantly
adopting a protective anti-inflammatory (M2) pheno-
type in the spinal cord of animal models of neurologi-
cal diseases such as amyotrophic lateral sclerosis [19]
or spinal cord injury [64]. In line with this observation,
our findings indicate that mice lacking xCT exhibit a dif-
ferent polarization profile of microglia as compared to
their wild-type counterparts, suggesting that the absence
of system xc− could beneficially balance the strong pro-
inflammatory activation state commonly observed in the
context of NP. Accordingly, an altered recruitment and
activation of glial cells in the spinal cord following nerve
lesion was observed after pharmacological inhibition of
system xc− with SAS. Together with a reduced astroglio-
sis and microgliosis, a reduced polarization toward the
pro-inflammatory microglial phenotype was observed.
is evidence aligns with the results of behavioral testing
indicating reduced pain sensitivity with SAS treatment as
compared to vehicle-treated animals.
From an experimental standpoint, our observations
highlight the need for complementary studies that would
(See gure on previous page.)
Fig. 5 Eect of sulfasalazine administration on the spinal expression of xCT and the pain hypersensitivity following PSNL surgery. Three (A) and seven (B)
days post-surgery, the ipsilateral and contralateral dorsal horn quadrants of the lumbar spinal cord of lesioned-xCT+/+ mice were dissected and used for
RT-qPCR. xCT mRNA was evaluated, normalized to the mean of 3 housekeeping genes (RPL-19, Ywhaz, HPRT-1) and expressed as a percentage of the re-
spective contralateral mRNA level. Pain hypersensitivity was assessed for the ipsilateral (C, D, F, G) and contralateral (E, H) hind paw at baseline (BL) and for
7 days following PSNL surgery. The 50% PWT to mechanical stimulation was used to determine allodynia (C-E), whereas the PWL to thermal stimulation
was used as a read-out of hyperalgesia (F-H). Panel D and G depict the area under the curve for the ipsilateral 50% PWT and PWL, respectively. Data are
presented as arithmetic mean with SEM (C, E, F, H) or geometric means with SD (D, G) (5 animal per group for mRNA analysis and 6 animal per group for
behavioral tests). Statistical analyses were performed using a one-way ANOVA (A, B) or a two-way ANOVA (D, G) model with repeated measures (C, E, F, H).
Main and interaction eects of the two-way ANOVA model are indicated below the graphs. When statistically signicant interaction was detected, Tukey’s
pairwise comparisons were conducted. P-values for signicant eects are reported on the graphs. In panel C and F, * indicates a signicant dierence
between SAS-treated wild-type (xCT+/+ SAS) and SAS-treated transgenic mice lacking xCT (xCT−/− SAS), # indicate a signicant dierence between SAS-
treated and saline-treated animals (within the same genotype). PSNL partial sciatic nerve ligation; PWT paw withdrawal threshold; PWL paw withdrawal
latency; AUC area under the curve; I ipsilateral; C contralateral; SAS sulfasalazine; G genotype eect; T treatment eect; G*T interaction eect
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Beckers et al. Journal of Neuroinammation (2024) 21:117
reinforce the therapeutic potential offered by blockers
of system xc−. With a focus on the implication of system
xc− during the onset of NP, the present study examined
animal behaviour and biochemical changes during the
first week after the surgical nerve lesion. Previous stud-
ies have shown that peripheral nerve lesions generate
long-lasting sensitization [65] and the implication of sys-
tem xc− in the persisting behaviour and associated bio-
chemical changes should also be examined at later time
points. Further studies could therefore consider initiat-
ing the administration SAS after the establishment of NP
to assess the role of system xc− in the pain maintenance.
is also underscores the opportunity of employing con-
ditional genetic models where xCT can be silenced at
later time points. Besides, it is worth mentioning that
the present findings were obtained using female mice
only, in order to reduce experimental variability. Hence,
pain hypersensitivity in models of chronic pain, includ-
ing from a neuropathic origin is sex-dependent and
further studies should include both males and females
[66, 67]. Finally, previous work has revealed that xCT-
deficient mice show reduced anxiety and depressive-like
Fig. 6 Eect of sulfasalazine administration on the spinal glial reactivity following PSNL surgery. Immunohistological staining of GFAP and Iba-1 was
performed on lumbar spinal cord samples of xCT+/+ mice at 3 (A, B, C) and 7 days (A, D, E) after surgery. The uorescent intensity for GFAP (B, D) and Iba-1
(C, E) in the dorsal horns was quantied. The results from the ipsilateral side are normalized and expressed as a percentage of the respective contralateral
side. Images shown are representative of 4 dierent animals per group and histograms represent the geometric means with SD. Statistical analyses were
performed using a one-way ANOVA model. GFAP glial brillary acidic protein; Iba-1 ionized calcium binding adapter protein 1; SAS sulfasalazine
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Beckers et al. Journal of Neuroinammation (2024) 21:117
Fig. 7 (See legend on next page.)
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Page 16 of 18
Beckers et al. Journal of Neuroinammation (2024) 21:117
behaviours, supporting a role for this exchanger in sev-
eral centrally controlled behaviours [68]. It is noteworthy
that anxiety and depression are comorbidities of chronic
pain and one could therefore anticipate a possible impact
of inactivating or inhibiting system xc− [69] on brain-
controlled behaviours associated with the neuropathic
lesion. In a broader perspective, further studies should
examine whether manipulating system xc− may influence
the processing of pain in supra-spinal centers.
Conclusion
Altogether, these findings provide strong evidence for
the important contribution of system xc− in the estab-
lishment of a persistent pathological pain state and spi-
nal neuroinflammation after peripheral nerve lesion. is
not only helps to better understand the molecular mech-
anisms supporting NP but offers new perspectives for
future therapies of this major clinical issue. Targeting sys-
tem xc− appears as a unique opportunity to tackle both
the altered glutamate transmission and the neuroinflam-
mation responsible for central sensitization in patients
suffering from NP. e pharmacological manipulation
of system xc− could therefore prevent, reduce, or allevi-
ate NP symptoms such as allodynia and hyperalgesia and
thereby improve the quality of life of affected patients.
Abbreviations
Arg-1 arginase 1
AUC area under the curve
CNS central nervous system
EAATs high-anity glutamate transporters
GFAP glial brillary acidic protein
HCA homocysteic acid
Iba-1 ionized calcium-binding adapter molecule 1
NOX-2 NADPH oxidase 2
NP neuropathic pain
PBS phosphate-buered saline
PSNL partial sciatic nerve ligation
PWL paw withdrawal latency
PWT paw withdrawal threshold
RT room temperature
RT-qPCR real-time quantitative polymerase chain reaction
SAS sulfasalazine
SEM standard error of the mean
Acknowledgements
Transgenic mice lacking xCT were generously provided by H. Sato
(Department of Medical Technology, Niigata University, Japan). We thank R.
Carvajal, F. Van der Kelen and the VUB-NAVI team for breeding the mice and
the excellent assistance for animal care. We would like to extend our gratitude
to François Beckers (from the Statistics Research Centre at the Katholieke
Universiteit Leuven, Belgium) for his expertise in statistical analyses.
Author contributions
Conceptualization of the study was primarily led by PB and EH. PB, MC and
ND performed the experiments. PB, MC and IB conducted the formal analysis
and contributed to the interpretation of the data. PB, IB, AM and EH wrote and
edited the manuscript. EH supervised the entire project. Funding acquisition
was managed by EH and AM. All authors provided critical feedback and
approved the nal version of the manuscript.
Funding
This work was supported by the Fonds Spéciaux de Recherche from the
Université catholique de Louvain, by the Belgium national funds for Scientic
research (FRS-FNRS grant Crédit aux chercheurs J.0106.24) and a Strategic
Research Program of VUB (SRP49 to Ann Massie).
Data availability
All data generated and analyzed during this study are included in this
published article.
Declarations
Ethics approval and consent to participate
All experiments were conducted in strict accordance with the
recommendations of the European commission and with the agreement of
the Belgian Ministry of Agriculture (code number LA 1230618). The ethical
committee of the Université catholique de Louvain (UCLouvain) for animal
experiments specically approved this study (aggregation number 2019/UCL/
MD/033).
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Author details
1Institute of Neuroscience, Group of Neuropharmacology, Université
catholique de Louvain (UCLouvain), Avenue Hippocrate 53 (B1.53.01),
Brussels 1200, Belgium
2Neuro-Aging & Viro-Immunotherapy, Center for Neurosciences, Vrije
Universiteit Brussel (VUB), Laarbeeklaan 103, Brussels 1090, Belgium
Received: 2 February 2024 / Accepted: 25 April 2024
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(See gure on previous page.)
Fig. 7 Eect of sulfasalazine on spinal mRNA of astrocytic and microglial markers after PSNL surgery in xCT+/+ mice. Three (left panels) and seven (right
panels) days post-surgery, the ipsilateral and contralateral quadrants of the lumbar spinal cord were dissected and used for RT-qPCR analysis. GFAP and
Iba-1 were used to evaluate astrocytic (A, B) and microglial (C, D) activation, respectively. Other markers were used to assess microglia activation prole
towards pro-inammatory (NOX-2: panel E & F) and anti-inammatory (Arg-1: panel G & H) phenotype. For all markers, the mRNA levels were normalized
to the mean of 3 housekeeping genes (RPL-19, Ywhaz, HPRT-1) and expressed as a percentage of the respective contralateral mRNA level. Data were
obtained from 5 dierent animals per group and are presented as geometric means with SD. Statistical analyses were performed using a one-way ANOVA
model. GFAP glial brillary acidic protein; Iba-1 ionized calcium binding adapter protein 1; NOX-2 NADPH oxidase 2; Arg-1 Arginase 1; SAS sulfasalazine
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

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