Brain, Behavior, and Immunity 95 (2021) 344–361

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Brain Behavior and Immunity

journal homepage: www.elsevier.com/locate/ybrbi

Microglial IL-10 and β-endorphin expression mediates gabapentinoids

antineuropathic pain
Khalil Ali Ahmad, Rana Muhammad Shoaib, Muhammad Zaeem Ahsan, Meng-Yan Deng, Le Ma,
Evhy Apryani, Xin-Yan Li, Yong-Xiang Wang *
King’s Lab, Shanghai Jiao Tong University School of Pharmacy, 800 Dongchuan Road, Shanghai 200240, China

A R T I C L E I N F O A B S T R A C T

Keywords: Gabapentinoids are recommended first-line treatments for neuropathic pain. They are neuronal voltage-
Gabapentinoids dependent calcium channel α2δ-1 subunit ligands and have been suggested to attenuate neuropathic pain via
Spinal cord interaction with neuronal α2δ-1 subunit. However, the current study revealed their microglial mechanisms
Neuropathic pain
underlying antineuropathic pain. Intrathecal injection of gabapentin, pregabalin and mirogabalin rapidly
Microglia
IL-10/β-endorphin
inhibited mechanical allodynia and thermal hyperalgesia, with projected ED50 values of 30.3, 6.2 and 1.5 µg (or
α2δ-1 176.9, 38.9 and 7.2 nmol) and Emax values of 66%, 61% and 65% MPE respectively for mechanical allodynia.
Intrathecal gabapentinoids stimulated spinal mRNA and protein expression of IL-10 and β-endorphin (but not
dynorphin A) in neuropathic rats with the time point parallel to their inhibition of allodynia, which was observed
in microglia but not astrocytes or neurons in spinal dorsal horns by using double immunofluorescence staining.
Intrathecal gabapentin alleviated pain hypersensitivity in male/female neuropathic but not male sham rats,
whereas it increased expression of spinal IL-10 and β-endorphin in male/female neuropathic and male sham rats.
Treatment with gabapentin, pregabalin and mirogabalin specifically upregulated IL-10 and β-endorphin mRNA
and protein expression in primary spinal microglial but not astrocytic or neuronal cells, with EC50 values of 41.3,
11.5 and 2.5 µM and 34.7, 13.3 and 2.8 µM respectively. Pretreatment with intrathecal microglial metabolic
inhibitor minocycline, IL-10 antibody, β-endorphin antiserum or μ-opioid receptor antagonist CTAP (but not κ- or
δ-opioid receptor antagonists) suppressed spinal gabapentinoids-inhibited mechanical allodynia. Immunofluo­
rescence staining exhibited specific α2δ-1 expression in neurons but not microglia or astrocytes in the spinal
dorsal horns or cultured primary spinal cells. Thus the results illustrate that gabapentinoids alleviate neuropathic
pain through stimulating expression of spinal microglial IL-10 and consequent β-endorphin.

1. Introduction
Gabapentin and pregabalin are anticonvulsant drugs that have been
prescribed as first-line treatments for neuropathic pain, including post­
herpetic neuralgia, peripheral diabetic neuropathy and fibromyalgia.
Their pain indications have been extended to postoperative pain,
headache, chronic low back pain and visceral pain (Stahl et al. 2013).
Their labeled and off-labeled pain uses, together with other indication
uses, have trebled in the United States over the past two decades
(González-Bueno et al. 2015), probably driven by raising awareness of
the importance of non-opioid pharmacologic therapies. Recently,
mirogabalin, a more potent analog, has also been approved in Japan to
treat peripheral neuropathic pain (Deeks 2019). Including gabapentin,
pregabalin and mirogabalin, gabapentinoids provide a major new class
of analgesics for neuropathic pain and pain treatment, in addition to
opioids and NSAIDs.
With the widespread use of gabapentinoids, it has been an active area
to figure out their molecular mechanisms underlying analgesia in
neuropathic pain. Gabapentin and pregabalin were first discovered to
bind to a high-affinity binding site in rat brain homogenates (Suman-
Chauhan et al. 1993), which was later found to be the α2δ-1 subunit of
voltage-gated Ca2+ channels (Gee et al. 1996). The voltage-gated Ca2+

Abbreviations: IL, interleukin; MOR, µ-opioid receptor; KOR, κ-opioid receptor; DOR, δ-opioid receptor; LPS, lipopolysaccharide; NASAIDs, non-ster­
oidal antiinflammatory drugs; CFA, complete Freund’s adjuvant; CRF, corticotropin-releasing factor; GABA, γ-aminobutyric acid; NMDA, N-methyl-D-aspartate; GLP-
1R, glucagon-like peptide-1 receptors; α7nAChRs, α7-nicotinic acetylcholine receptors; GPR40, G-protein receptor 40; PBS, phosphate-buffered saline.
* Corresponding author.
E-mail address: yxwang@sjtu.edu.cn (Y.-X. Wang).

https://doi.org/10.1016/j.bbi.2021.04.007
Received 27 December 2020; Received in revised form 11 April 2021; Accepted 12 April 2021
Available online 20 April 2021
0889-1591/© 2021 Elsevier Inc. All rights reserved.
K.A. Ahmad et al.
channels (particularly N-type calcium channels) are associated with the
two auxiliary α2δ-1 and β subunits, and their coexpression with the main
pore-forming α1 subunit leads to a significant rise in the cell Ca2+ cur­
rent amplitude and a role in Ca2+ channels membrane incorporation.
Neuronal α2δ-1 has been accepted to be the target site for gabapenti­
noids (Cheng and Chiou, 2006; Stahl et al., 2013), with binding affinities
(Kd) in nanomolar (Domon et al., 2018a, 2018b; Li et al., 2011; Suman-
Chauhan et al., 1993). α2δ-1 is expressed predominantly in spinal dorsal
horn neurons, localized in the superficial laminae and moderately
expressed postsynaptically on deeper neurons (Li et al., 2006; Bauer
et al., 2009). It was discovered that peripheral nerve injury upregulated
presynaptic α2δ-1 expression and trafficking, which accounted for
neuropathic pain development (Bauer et al., 2009; Hoppa et al., 2012;
Zhou and David, 2013). It has been suggested that gabapentin and
pregabalin interfere with anterograde trafficking of α2δ-1 from dorsal
root ganglion neurons to the presynaptic terminals of the dorsal horns,
resulting in reduced release of pain neurotransmitters such as glutamate
and spinal sensitization, and subsequently attenuate neuropathic pain
(Stahl et al., 2013; Hendrich et al., 2008). However, the gabapentinoids
results are inconsistent with inhibition of Ca2+ currents, pain transmitter
release and α2δ-1 upregulation and trafficking (Bauer et al., 2010; Chen
et al., 2018; Bannister et al., 2011; Yang et al., 2014). There is also
discrepancy between gabapentinoids binding with high affinities to α2δ-
1 subunit (in nanomolar range) and their low potencies in physiological
actions (in low micromolar range), particularly clinical analgesic dosage
up to 3,600 mg for gabapentin (Stahl et al. 2013). In addition, alteration
of α2δ-1 upregulation and trafficking is associated with chronic appli­
cation (days) of gabapentinoids, while analgesia in pain hypersensitivity
effects relatively rapid (within 30 min of gabapentinoids administration)
(Yang et al., 2012; Bauer et al., 2009; Chen et al., 2018). Thus gaba­
pentinoids may exert antineuropathic pain by other mechanisms.
Indeed, other receptor targets have also been suggested, including so­
dium channels, hyperpolarization-activated cation channels and
particularly α2δ-1-NMDA receptor complexes (Chen et al., 2018; Tae
et al., 2017; Yang et al., 2009).
Historically, only neurons were believed to be involved in the pa­
thology of neuropathic pain. However, microglia and microglia-neuron
interactions have been associated with initial and maintenance of syn­
aptic plasticity and central sensitization after peripheral nerve injury in
recent years (Tiwari et al., 2014; Scholz and Woolf, 2007; Ji et al.,
2013). In contrast to microglia activation (classic role) which releases
neuroinflammatory factors such as interleukin (IL)-6, IL-1β, tumor ne­
crosis factor (TNF)-α and brain-derived neurotrophic factor (BDNF), and
subsequently provokes spinal dorsal horn neurons (Kawasaki et al.
2008), alternatively activated microglia release antiinflammatory fac­
tors such as IL-10, IL-4, nerve growth factor (NGF), glial cell line-derived
neurotrophic factor (GDNF), and endogenous opioid peptides have been
recently identified to provide tissue repairing, neuroprotective and
analgesic actions in neuropathic pain (Wu et al., 2018a; Li et al., 2016;
Huang et al., 2016; Franco and Fernandez-Suarez, 2015; Carniglia et al.,
2017). It has been reported that Aconitum-derived bulleyaconitine A,
bullatine A, lappaconitine and isotalitizindine induced pain anti­
hypersensitivity through microglial dynorphin A expression (Shao et al.,
2020; Huang et al., 2020, 2016; Li et al., 2016; Sun et al., 2018). Also, IL-
10 administered intrathecally to neuropathic rats yielded remarkable
inhibition of mechanical allodynia and thermal hyperalgesia through
spinal microglial β-endorphin expression (Wu et al. 2018a). Interest­
ingly, this IL-10/β-endorphin pathway accounts for pain relieve effects
caused by agonism of the GLP-1Rs, α7nAChRs, and GPR40 in numerous
rodent pain models (Apryani et al., 2020; Mao et al., 2019; Wu et al.,
2017b).
Gabapentinoids analgesia has been recently implicated in microglia,
although the findings were somewhat controversial. Gabapentin was
reported to inhibit allodynia and hyperalgesia in rat models of CFA-
induced chronic myositis and monoarthritis and streptozotocin-
induced diabetes, which was associated with inhibition of microglial
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activation (Rosa et al., 2017; Wodarski et al., 2009). On the other hand,
gabapentin was reported to exert antinociception in neuropathic rats via
upregulation of spinal antiinflammatory cytokine IL-10 and subsequent
inhibition of proinflammatory cytokines containing TNF-α, IL-6 and IL-
1β (Lee et al. 2013). By the same mode, the intrathecal administration of
gabapentin also enhanced morphine antinociception (Bao et al. 2014b).
Thus the present study intended to investigate the microglial mecha­
nisms underlying gabapentinoids analgesia in neuropathic pain, mainly
the relationship with the spinal IL-10/β-endorphin microglial signaling
pathway. Firstly, we evaluated the inhibitory effects of intrathecal
gabapentin, pregabalin and mirogabalin on mechanical allodynia in a
neuropathic pain model. Then we determined whether gabapentinoids
specifically expressed IL-10 and β-endorphin in spinal microglia. Addi­
tionally, we directly measured gabapentinoids on IL-10 and β-endorphin
expression in microglial cells. Further, we explored the causal rela­
tionship between their analgesia and microglial IL-10 and β-endorphin
expression. Lastly, we made efforts to identify molecule targets for
gabapentinoids to express IL-10 and β-endorphin in microglia. Our re­
sults demonstrated that spinal gabapentinoids produced fast anti­
neuropathic effects through microglial IL-10 and β-endorphin
expression.
2. Materials and methods
2.1. Drugs and chemicals
Gabapentin and pregabalin were gifts from Enhua Pharmaceuticals
(Xuzhou, Jiangsu, China) and mirogabalin was purchased from Med­
ChemExpress (NJ, USA). Minocycline hydrochloride and lidocaine hy­
drochloride were obtained from Yuanye Biotech (Shanghai, China) and
Chengdu Pharmaceuticals Group (Chengdu, China) respectively, while
DPCPX and GNTI were acquired from Sigma-Aldrich (USA). CTAP,
naltrindole and β-endorphin polyclonal antiserum were bought from
Abcam (Cambridge, UK), and recombinant rat-IL-10 antibody was from
R&D Systems (USA). α-helical CRF(9–41) and Exendin9-39 were
commercially synthesized by GL Biochem (Shanghai, China) and
Shanghai TASH Biotechnology Co. (China) respectively, with peptide
contents of 98%. GW1100 and α-helical CRF(9–41) were dissolved in
70% polyethylene glycol (PEG400) and30% dimethyl sulfoxide (DMSO)
in 0.9% normal saline (Mao et al., 2019; Apryani et al., 2020), while
DPCPX was suspended in 10% DMSO in 0.9% normal saline.
2.2. Experimental animals
One-day-old (sex unidentified) neonatal Wistar rats and male or fe­
male adult rats (180–200 g) were acquired from Institute of Shanghai
Experimental Animals (China). Adult rats were kept in specific plastic
boxes (4 per cage) at typical room temperature and humidity on 12 h
dark / light cycles in the Central Animal Laboratory, under ad libitum
feeding. The animals were adapted for 3 to 5 days for laboratory at­
mosphere before operations or experiments. Experimental study groups
were allocated randomly, and the researchers were blinded to the
behavior tests. The Animal Care and Welfare Committee of Shanghai
Jiao Tong University (Shanghai, China) approved the animal research
protocols, which performed according to the international animal care
guidelines (National Institutes of health, USA).
2.3. Primary neuronal and glial cells cultures
Neuronal and glial cells were obtained from the spinal cords of 1-
day-old neonatal rats. The extracted spinal cords were digested with
0.05% trypsin. The cells were disassociated and then suspended in
DMEM supplemented with 10% (v/v) fetal bovine serum (FBS), strep­
tomycin (100 μg/mL), and penicillin (100 U/ml). For neuronal culture,
the cells were then incubated for 1.5 h in 24-well cell culture plates in
the complete DMEM. The culture medium was then changed to the
K.A. Ahmad et al.
Neurobasal (Invitrogen) containing B27 and 0.5 mM glutamine for
further culture. All experiments were started after 6 day of plating.
Harvested neurons showed a purity > 85% as determined by the NeuN
immunoreactivity.
For glial cell cultures, cells suspension was incubated in pre-coated
(poly-L-lysine, 100 µg/ml) 75-cm2 cell culture flasks (1 × 107 cells/
flask) and maintained at 37 ◦ C in a 5% carbon dioxide cell incubator. To
prepare microglial cells, after 8 days the aliquots were obtained by
shaking the cultural flasks at 260 rpm, 37 ◦ C for 2 h. The cells suspension
was then plated in new plates and unattached cells were removed by
washing with serum-free DMEM. Harvested microglial cells showed a
purity > 95%, as determined by the Iba-1 immunoreactivity.
To prepare astrocyte cells, after 11 days culture the flasks were also
shaken for 2 h followed by the incubation with 10 mL of 0.05% trypsin-
EDTA (Invitrogen) in a cell incubator for 15 min to separate the oligo­
dendrocytes from astrocytes. After the trypsin neutralization by 10 mL
of the complete DMEM medium, the floating cells suspension was dis­
carded. A nearly intact bed layer of astrocyte cells was then subcultured
conventionally in new plates after trypsinization. Cultured astrocytes
showed > 90% purity as verified by the GFAP immunoreactivity.
2.4. Intrathecal catheterization and injection in rats
A polyethylene catheter (PE-10: 0.28 mm inner diameter and 0.61
mm outer diameter; Clay Adams, Parsippany, NJ, USA) was implanted
into the rat’s spinal cord lumbar level under standard inhaled isoflurane
anesthesia run by an anesthesiameter (Ugo Basile Gas Anesthesia Sys­
tem) as described previously (Huang et al., 2012; Lu et al., 2012). The
precise catheter position was validated by injecting 4% lidocaine (10 μL)
2–3 days after recovery. Rats which showed no motor impairments, for
example paralysis and claudication following implanting of the catheter
and showed instant bilateral paralysis of the hindlimbs following
intrathecal injection of lidocaine were included for additional experi­
mental research. For intrathecal delivery, 10 μL of drug solution (fol­
lowed by a 15-μL saline for flushing) was injected using a 50-μL
microliter syringes (Shanghai Anting Micro-Injector Factory, Shanghai,
China).
2.5. Neuropathic pain rat model and behavior assessment
The rats were undergone to L5/L6 unilateral spinal nerve ligation
(SNL) to produce peripheral neuropathy basically as described previ­
ously (Kim and Chung, 1992). Briefly, rats L5 and L6 spinal nerves were
carefully uncovered and ligated with 6–0 silk thread tightly under
inhaled isoflurane anesthesia. After 2 spinal nerves ligated, the lumbar
fascia was sutured by absorbable suture in layers and the skin was
stitched. Rats were allocated to retrieve afterwards, and only rats which
showed substantial ipsilateral paws mechanical allodynia to inoffensive
mechanical stimulus (hindlimbs withdrawal threshold in the ipsilateral
surgery side < 8 g) and without motor- reflex shortages were selected for
the additional research tests. Sham rats underwent the same surgical
procedures described above except without spinal nerves ligation.
To assess the mechanical allodynia, rats were firstly adapted to
testing environment for 30 min, specifically on a metal grid in trans­
parent plexiglass boxes. The contralateral and ipsilateral hindlimb
withdrawal threshold was evaluated using an electronic Von Frey
monofilament (2450 CE, USA). The hand held probe contacted to a
monofilament No.15 generated force (0.1 to 90 g) was used to stimulate
base of the hindpaws. The threshold was considered as the least force to
evoke the withdrawal response of the rat’s hindpaws. The mean of
triplicate readings at 5 min intervals was recorded for each hindlimb at
each time point (Li et al. 2016).
The Hargreaves test with heated glass (IITC Life Science Inc.) was
used to assess thermal hyperalgesia. Briefly, rats were acclimatized for
the testing environment by placing them in a plexiglass box for at least
thirty minutes. The radiant heat source was applied on the plantar
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Brain Behavior and Immunity 95 (2021) 344–361
medial surface of each hindpaw. The hindpaw withdrawal latency was
considered as the duration (seconds) of time from the start of radiant
heat application till a sudden withdrawal response. The cut-off of 30 s
was set to avoid tissue damage. Each hindpaw was tested independently
in triplicate with five minutes intervals. The final results were computed
from the mean of three independent measurements.
2.6. RNA extraction and real-time qPCR
Spinal lumbar enlargements from sham or neuropathic rats were
collected and homogenized mechanically by an electronic micro-
homogenizer (4,000 rpm for 15 s) in TRIzol (Invitrogen, Thermo­
fisher). The primary cultured cell samples were also obtained and lysed
in TRIzol after drug treatment. The amount of 1 µg total RNAs were then
transcribed reversely into cDNA using a ReverTra Ace qPCR RT kit
following the manufacturer protocols (Toyobo Co., Osaka, Japan). Real-
time qPCR was afterwards performed by utilizing the RealmasterMix
SYBR Green I (Yeasen Biotechnology) in a Mastercycler ep realplex
(Eppendorf, Hamburg) in mixture with either of the following specific
primers: 5′ -CCAAGGTCATCCATGACAAC-3′ and 5′ -TCCA­
CAGTCTTCTGAGTGGC-3′ for GAPDH (Gong et al. 2014), 5′ -
GGCTCAGCACTGCTATGTTGCC-3′ and 5′ -AGCATGTGGGTCTGGCT­
GACTG-3′ for IL-10 (Wu et al. 2017b), 5′ -CCTATCGGGTGGAGCACTTC-
3′ and 5′ -TGGCTCTTCTCGGAGGTCAT-3′ for the β-endorphin precursor
proopiomelanocortin (POMC) (Wu et al. 2018a), and 5′ -
CCTGTCCTTGTGTTCCCTGT-3′ and 5′ -AGAGGCAGTCAGGGTGAGAA-3′
for the dynorphin precursor prodynorphin (PDYN) (Huang et al. 2016).
Real-time qPCR was firstly used to verify the primers specificity through
the melting curves and the relative mRNA expression of the targeted
genes was then determined by the (2 − ΔΔCt) method after normali­
zation to the housekeeping gene GAPDH (cycle threshold) values.
2.7. Immunofluorescence staining
The co-labeling immunostaining of IL-10, β-endorphin, α2δ-1, and/
or microglial, astrocytic and neuronal cellular biomarkers in frozen
sections of spinal cords and primary culture cells were performed and
pictured under a confocal microscope (TCS SP8, Leica Microsystems,
Wetzlar, Germany) as described previously (Huang et al. 2016). Sham or
neuropathic rats were undergone to anesthetize by injection of pento­
barbital (50 mg/kg, intraperitoneal) and intracardially perfused with
normal saline (100 mL) and then 4% paraformaldehyde (PFA, 60 mL).
The lumbar enlargements (L3–L6) were carefully dissected and fixed
overnight at 4 ◦ C in 4% buffered PFA. Tissues were then dehydrated
gradually in 10%, 20% and 30% sucrose solutions at 4 ◦ C. Frozen sec­
tions were prepared in OCT embedding media (Leica Microsystems) and
sliced into 30-μm thickness. Spinal glial and neuronal cells were cultured
in 24-well plates (5 × 104 cells/well) on coverslips and then fixed by 4%
buffered PFA. Both cell coverslips and frozen sections were blocked by
incubation for 1 h in goat serum (10%, v/v) and Triton X-100 (0.5%, v/
v) in PBS. Cell coverslips and frozen sections were kept with the specific
primary antibodies: rat IL-10 antibody (1:200; rabbit polyclonal; Pro­
teinTech Group, IL, USA), rat α2δ-1 antibody (1:200; rabbit polyclonal;
ProteinTech Group) and rat β-endorphin antibody (1:200; rabbit poly­
clonal; Phoenix Pharmaceuticals, USA), as well as antibodies against
primary cellular biomarkers for overnight at 4 ◦ C. Spinal glial and
neuronal cells were observed with the following biomarkers: NeuN
(1:100 for frozen sections or 1:200 for cell coverslips, mouse polyclonal;
Millipore, Darmstadt, Germany) for neurons, Iba-1 (1:100 for frozen
sections or 1:200 for cell coverslips, Millipore) for microglia and GFAP
(1:200 for frozen sections and cell coverslips, mouse polyclonal; Milli­
pore) for astrocytes. IL-10, β-endorphin and α2δ-1 were observed by the
following secondary antibody, Alexa-555-conjugated goat anti-rabbit
(1:200, Abcam). Other primary antibodies were visualized by the sec­
ondary antibody, Alexa-488-conjugated goat anti-mouse (1:200,
Abcam). For cell coverslips, the 49,6-diamidino-2-phenylindole (DAPI,
K.A. Ahmad et al.
Fig. 1. Inhibitory effects of intrathecal gabapentin (A), pregabalin (B) and mirogabalin (C) on mechanical allodynia of in neuropathic rats. Dose-response curves of
gabapentin (D), pregabalin (E) and mirogabalin (F) on mechanical allodynia 1 h after injection. Inhibitory effects of intrathecal gabapentinoids on thermal anti­
hyperalgesia in neuropathic rats (G). The data are presented as means ± SEM (n = 6 per group).
Beyotime Biotechnology, Shanghai, China) staining was applied to the
cell nuclei.
To quantify the IL-10-, β-endorphin-, α2δ-1-, NeuN-, GFAP-, and Iba-
1-immunopositive cell, photomicrographs of the medial three fourths of
the dorsal horns (laminas I-V) were captured by the confocal microscope
at 10 × or 40 × magnifications. A researcher who was known the
experimental groups calculated the positive stained regions using
ImageJ Software (National Institutes of Health, USA). Background
fluorescence was excluded and only positive co-labeling from the cell
surfaces was included by the high and low threshold set up. The same
ImageJ software settings was applied for all control and treated groups.
The mean percentage of the co-labeled areas was the positive fraction of
the total measured image region from 3 non-adjacent slices of every
spinal cord (Huang et al., 2016; Wu et al., 2017b).
2.8. Western blot
Western blot was performed as described previously by (Fan et al.
2016). Briefly, the spinal lumbar enlargements were homogenized and
proteins were extracted using the radio-immunoprecipitation assay
buffer containing 1% protease inhibitor phenylmethylsulfonyl fluoride.
The lysates were centrifuged for 15 min at 12,000 rpm at 4 ◦ C. The
protein concentration of samples was measured using the bicinchoninic
acid assay (Beyotime Biotechnology). The proteins were separated by
10% sodium dodecyl sulfate–polyacrylamide gel electrophores and then
transferred to polyvinylidene fluoride membranes by an electrophoresis
technique. The membranes were blocked in 5% skim milk powder in
Tris-based saline containing 0.1% Tween 20 at room temperature for 1
h, and then incubated with primary antibodies raised against the α2δ-1
subunit (1:1000, #27452–1-AP, rabbit polyclonal, ProteinTech Group,
USA) and GAPDH (1:5000, #6004–1-Ig, mouse polyclonal, ProteinTech
Group, USA) overnight at 4 ◦ C with gentle shanking. Protein bands were
visualized using the Odyssey Infrared Imaging system (Li-Cor Bio­
sciences, USA) after 1-hour incubation at room temperature with the
goat anti-rabbit IgG, Dylight 800-conjugate (1:10,000, #5151, Cell
Signaling Technology Inc. USA) or goat anti-mouse IgG, Dylight 680-
conjugate (1:10,000, #5470, Cell Signaling Technology Inc. USA).
Bands intensities were quantitated using the image analysis software
(ImageJ Software, National Institutes of Health). The relative expression
of the target protein was calculated after normalization to the GAPDH
level. The experiments were repeated at least treble.
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Brain Behavior and Immunity 95 (2021) 344–361
2.9. β-Endorphin , dynorphin a and IL-10 measurement
The concentrations of β-endorphin, dynorphin A and IL-10 were
measured in the spinal lumbar enlargements from sham or neuropathic
rats 1 h after intrathecal drug treatments. The spinal lumber enlarge­
ments supernatants collected after homogenizing (4,000 rpm for 15 s) in
10 mM Tris-hydrochloric acid (pH 7.4) and then centrifuged (4000 rpm
and 4 ◦ C) for 15 min. Protein concentrations were also measured by the
standard method, bicinchoninic acid test following the commercial kit
instructions (Beyotime Biotechnology). β-endorphin or IL-10 concen­
trations were detected in neuronal, astrocytic, and microglial culture
cells. Cells were plated in 24-well culture plates (5 × 105 cells per well)
and gently washed before drug treatments with warm DMEM (1 mL)
containing bovine serum albumin (2 mg/mL) and N-(2-hydroxyethyl)
piperazine-N0-2-ethanesulfonic acid (HEPES, 15 mmol/L). Cells were
then treated with the experimental drugs for 2 h and sample superna­
tants were preceded. According to the manufacturer instructions, spe­
cific enzyme-linked fluorescent immunoassay kits were used to measure
the β-endorphin, dynorphin A (Phoenix Pharmaceuticals) and IL-10
(eBioscience) concentrations which were validated by running stan­
dard curves with the testing samples simultaneously to determine the
unknown concentrations. The relative fluorescence units were detected
by a Microplate Reader (ThermoLab systems, Finland) and the IL-10,
β-endorphin or dynorphin A concentrations were determined by com­
parisons with the calibration curves performed simultaneously.
2.10. Data calculation and statistical evaluation
To compute percentage of the maximal possible effect (% MPE) the
following formula was applied: (post-drug threshold in ipsilateral hin­
dlimb – baseline threshold in ipsilateral hindlimb) / (baseline threshold
in contralateral hindlimb –baseline threshold in ipsilateral hindlimb) ×
100. The % MPE values near to 100 were regarded as normal mechanical
thresholds in the contralateral hindpaws, while values closed to 0 indi­
cate mechanical allodynia in the ipsilateral hindpaws. For analysis of the
dose–response curves, the parameters, i.e., the minimum effect,
maximum effect (Emax), half-effective concentration or dose (EC50 or
ED50), and Hill coefficient (n), were calculated by fitting nonlinear least-
squares curves to the relation Y = a + bx, where × = [C]n/(ECn50 + [C]n)
or [D]n/(EDn50 + [D]n). The values of EC50 or ED50 and b (Emax) were
projected by yielding a minimum residual sum of squares of deviations
from the theoretical curve (Wang and Pang 1993).
K.A. Ahmad et al.
Fig. 2. Effects of gabapentinoids intrathecally on IL-10, POMC, PDYN mRNA expression (A-C), and IL-10, β-endorphin and dynorphin A protein expression (D-F) in
neuropathic rats. Spinal lumbar enlargements were obtained 1 h after injection. Data are shown as means ± SEM (n = 6 per group). * P < 0.05 compared with the
control group, by one-way ANOVA followed by the post-hoc Bonferroni test.
Data are summarized as means ± SEM or 95% confidence limits. The
significance of the differences was calculated by unpaired and two-way
Student t-test or one-way or repeated-measures two-way ANOVA using
GraphPad Prism (version 8.0.1, GraphPad Software, Inc., USA). The
post-hoc Bonferroni test was used when the effect of the drug (dose) (for
the one-way ANOVA, the factor was drug [dose]; for the two-way
ANOVA, the factors were drug [dose], time, and their interaction) was
statistically significant. Probability values were two-tailed and the sta­
tistical significance criterion p-value was 0.05.
3. Results
3.1. Gabapentinoids reduced mechanical allodynia and thermal
hyperalgesia in neuropathic rats
A single injection of normal saline (10 µL) or gabapentin (3, 10, 30,
100 or 300 µg), pregabalin (1, 3, 10, 30 or 100 µg) or mirogabalin (0.3,
1, 3, 10 or 30 µg) administrated intrathecally to six groups of neuro­
pathic rats. The paw withdrawal responses were afterwards measured
with 5-min intervals before, and 0.5, 1, 2, and 4 h after the intrathecal
drug injection. As shown in Fig. 1A-1C, spinal nerve ligation remarkably
provoked mechanical allodynia in the ipsilateral hindpaws, compared
with the contralateral hindpaws. After intrathecal administration of
normal saline, hindpaw withdrawal responses in neuropathic rats
remained unchanged. Whereas intrathecal injection of gabapentinoids
augmented withdrawal thresholds dose- and time-dependent in the
ipsilateral hindpaws, with peak effect at 1 h after injection (P < 0.05, by
repeated-measures two-way ANOVA followed by the post-hoc Bonfer­
roni test), without affecting withdrawal thresholds in the contralateral
hindpaws. The dose–response analyses were computed from the % MPE
values of each drug obtained 1 h after injection. The ED50 and Emax
values projected for gabapentin, pregabalin and mirogabalin were 30.3
µg or 176.9 nmol (95% confidence limits: 21.9 to 41.9 µg or 127.9 to
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Brain Behavior and Immunity 95 (2021) 344–361
244.7 nmol) and 66% MPE, 6.2 µg or 38.9 nmol (95% confidence limits:
4.8 to 7.8 µg or 30.1 to 49.0 nmol) and 61% MPE, and 1.5 µg or 7.2 nmol
(95% confidence limits: 1.1 to 2.0 µg or 5.2 to 9.6 nmol) and 65% MPE,
respectively (Fig. 1D-1F). Gabapentinoids-inhibited thermal hyper­
algesia was also assessed in neuropathic rats. Four groups of SNL rats
received intrathecal injection of saline (10 µL), gabapentin (100 µg),
pregabalin (30 µg) or mirogabalin (10 µg). Hindpaw withdrawal la­
tencies were measured with 5-min intervals before, and 0.5, 1, 2, and 4 h
after intrathecal drug injection. As shown in Fig. 1G, spinal nerve injury
induced notable thermal hyperalgesia in the ipsilateral hindpaws
compared to the contralateral hindpaws. Intrathecal saline injection (10
µL) did not influence paw withdrawal latencies in both hindpaws.
However, gabapentin, pregabalin and mirogabalin significantly inhibi­
ted thermal hyperalgesia in the ipsilateral hindpaws (P < 0.05, by
repeated-measures two-way ANOVA followed by the post-hoc Bonfer­
roni test), without affecting withdrawal latencies in the contralateral
hindpaws.
3.2. Gabapentinoids specifically stimulated spinal microglial IL-10 and
β-endorphin expression
To assess the effects of gabapentinoids in the expression of spinal IL-
10, β-endorphin and dynorphin A, four groups of neuropathic rats were
injected intrathecally with normal saline (10 μL), gabapentin (100 μg),
pregabalin (30 μg) or mirogabalin (10 μg), and spinal lumber enlarge­
ments were isolated 1 h after intrathecal administration. Measured by
RT-qPCR, gabapentinoids considerably increased the mRNA IL-10 by
80% (Fig. 2A) and POMC expression by 77% (Fig. 2B), but failed to
significantly up-regulate the gene expression of PDYN (Fig. 2C). Also,
the effects of gabapentinoids in the IL-10, β-endorphin and dynorphin A
expression were further assessed by commercial fluorescent immuno­
assay kits. The baseline protein levels of IL-10, β-endorphin and
dynorphin were 6.4 ± 1.9, 22.5 ± 2.4 and 20.6 ± 3.8 pg/mg respectively
K.A. Ahmad et al. Brain Behavior and Immunity 95 (2021) 344–361

Fig. 3. Stimulatory effects of gabapentin administered intrathecally on spinal IL-10 microglial expression in neuropathic rats. Co-labeling immunostaining of IL-10
antibody with microglial (Iba-1) marker, astrocytic (GFAP) marker or neuronal (NeuN) marker in spinal dorsal horn (scale bar: 250 µm) and laminae I-V (scale bar:
50 µm, A-F). Yellow co-labeling of IL-10 and cellular markers shown by arrows. G-L. Stimulatory effect of gabapentin. The % immunolabeled surface areas of IL-10/
Iba-1 (M), IL-10/GFAP (N) and IL-10/NeuN (O) from the spinal dorsal horn laminae I-V were quantified using ImageJ software. Data are presented as means ± SEM
(n = 6 per group). * P < 0.05 compared with control group by unpaired and two-tailed Student t-test. (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)

in the spinal homogenates from the saline treated neuropathic rats.
Gabapentinoids intrathecally significantly increased expression of IL-10
by 165% (Fig. 2D) and β-endorphin by 86% (Fig. 2E) but not dynorphin
A (Fig. 2F).
To determine the spinal cell types that specifically elevated IL-10
expression after gabapentin intrathecal treatment, double label immu­
nostaining was applied by co-labeling of IL-10 with the cellular bio­
markers of neurons (NeuN), astrocytes (GFAP), and microglia (Iba-1) in
spinal frozen sections of neuropathic rats. Two groups received normal
saline (10 μL) or gabapentin (100 μg) intrathecally and the spinal lumber
enlargements were obtained 1 h after injection. As shown, IL-10 staining
was co-localized widely with Iba-1 (Fig. 3A, 3B) of ipsilateral spinal
dorsal horns (I-V laminae), but less with the GFAP (Fig. 3C, 3D) or NeuN
(Fig. 3E, 3F). Gabapentin (100 µg) intrathecally stimulated IL-10 im­
munostaining with Iba-1 (Fig. 3G, 3H), but did not stimulate the co-
labeling of IL-10/GFAP (Fig. 3I, 3 J) or IL-10/NeuN (Fig. 3K, 3L). The
double immunofluorescences staining of IL-10 and cellular biomarkers
were quantitatively measured to calculate immunolabeled area fractions
in spinal dorsal horns (laminae I-V) by ImageJ Software. Compared with
saline groups, gabapentin significantly increased the co-labeling im­
munostaining of IL-10/Iba-1 by 2.9-fold, but not IL-10/GFAP (Fig. 3N)
or IL-10/NeuN (Fig. 3O).
Additionally, we also determined the specific-cell types which
elevated the β-endorphin expression after gabapentin treatment. Co-
labeling of β-endorphin with NeuN, GFAP and Iba-1 was performed in
the spinal frozen sections from the aforementioned two groups of
neuropathic rats. β-Endorphin labeling was co-localized with Iba-1
(Fig. 4A, 4B), GFAP (Fig. 4C, 4D) NeuN (Fig. 4E, 4F) in the ipsilateral
dorsal horns (I–V laminae) of the neuropathic rats. Gabapentin mark­
edly promoted co-labeling of β-endorphin/Iba-1 (Fig. 4G, 4H), but not
β-endorphin/GFAP (Fig. 4I, 4 J) or β-endorphin/NeuN (Fig. 4K, 4L).
Quantitatively, gabapentin treatment augmented co-labeling of
β-endorphin/Iba-1 by 3.1-fold as measured by ImageJ Software
(Fig. 4M), but not β-endorphin/ GFAP (Fig. 4N) or β-endorphin/NeuN
(Fig. 4O).
To compare gabapentinoids-induced antihypersensitivity and stim­
ulation of spinal IL-10 and β-endorphin expression, six groups of male
sham rats and male and female SNL rats were intrathecally injected with
saline (10 µL) or gabapentin (100 µg). One hour later, the hindpaw
withdrawal thresholds or latencies were measured in the ipsilateral
hindpaws. As shown in Fig. 5A and 5B, compared to saline treatment,
intrathecal gabapentin failed to produce significant changes in with­
drawal thresholds or latencies in male sham rats. In contrast, gabapentin
inhibited mechanical allodynia and thermal hyperalgesia in both male
and female SNL rats by nearly the same degrees. Rats were immediately
killed and spinal lumber enlargements were collocated after behavioral
tests for IL-10 and β-endorphin gene and protein quantification.
Compared to saline control, intrathecal gabapentin (100 µg) signifi­
cantly increased IL-10 mRNA expression by 60%, 76% and 78%, and
POMC mRNA expression by 68%, 71% and 72% in male sham rats, male
and female SNL rats, respectively (Fig. 5C, 5D). In addition, treatment
with gabapentin significantly increased IL-10 protein expression by
140%, 165% and 160%, and β-endorphin protein expression by 66%,
86% and 89% in these animals, respectively (Fig. 5E, 5F). Gabapentin-
stimulated microglial IL-10 expression was further compared using the
immunostaining technique. Frozen sections from the ipsilateral spinal
cords were co-labeled with the IL-10 antibody and microglial biomarker
Iba-1 antibody. As shown in Fig. 5G, intrathecal gabapentin remarkably
increased co-immunostaining of IL-10/Iba-1 in male sham, and male and
female SNL rats. Quantitatively by using the ImageJ computer software,
gabapentin-increased IL-10/Iba-1 colocalization in these groups of rats
was 140%, 150% and 149%, respectively (Fig. 5H).

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Fig. 4. Stimulatory effects of gabapentin administered intrathecally on spinal β-endorphin microglial expression in neuropathy rats. Co-labeling immunostaining of
β-endorphin antibody with microglial (Iba-1) marker, astrocytic (GFAP) marker or neuronal (NeuN) marker in spinal dorsal horn (scale bar: 250 µm) and laminae I-V
(scale bar: 50 µm, A-F). Yellow co-labeling of β-endorphin and cellular markers shown by arrows. G-L. Stimulatory effect of gabapentin. The % immunolabeled
surface areas of β-endorphin/Iba-1 (M), β-endorphin/GFAP (N), and β-endorphin/NeuN (O) from spinal dorsal horn laminae I-V were quantified using ImageJ
software. The data are shown as means ± SEM (n = 6 per group). * P < 0.05 compared with the control group by unpaired and two-tailed Student t-test. (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

3.3. Gabapentinoids specifically stimulated spinal microglial IL-10 and
β-endorphin expression
To additionally verify the specific stimulatory effects of gabapenti­
noids on spinal microglial IL-10 and POMC gene expression, primary
microglial culture cells were incubated with vehicle, gabapentin, pre­
gabalin and mirogabalin for 2 h. The cellular IL-10 and POMC mRNA
expression were measured by RT-qPCR. Treatment with gabapentin
(100 µM), pregabalin (30 µM) and mirogabalin (10 µM) promoted the IL-
10 gene expression of microglial, but not astrocytic or neuronal cells
(Fig. 6A-6C). The same specific stimulatory effects of gabapentinoids on
POMC expression were also observed in primary microglial but not
astrocytic or neuronal cells (Fig. 6D-6F). Further dose–response analysis
indicated that gradient concentrations of gabapentin (3, 10, 30,100 and
300 µM), pregabalin (1, 3, 10, 30 and 100 µM) and mirogabalin (0.3, 1,
3, 10 and 30 µM) concentration-dependently upregulated the mRNA
expression of IL-10 and POMC in primary microglia with EC50 values of
41.3, 11.5 and 2.5 µM and 34.7, 13.3 and 2.8 µM, respectively, both of
which were overlapped (Fig. 6G-6I).
The stimulatory effects of gabapentinoids on the IL-10 and
β-endorphin levels in primary microglial cultured cells were also
compared with primary spinal astrocytes and neurons using commercial
fluorescent immunoassay kits. Treatment with the vehicle, gabapentin
(100 µM), pregabalin (30 µM) and mirogabalin (10 µM) for 2 h specif­
ically increased the IL-10 levels by approximately 3-fold (Fig. 7A-7C)
and β-endorphin by approximately 2.8-fold (Fig. 7D-7F) in the culture
medium of microglial cells, but not of astrocytes or neurons.
To further ascertain the stimulatory effect of gabapentin on micro­
glial IL-10 and β-endorphin expression, the microglial inhibitor mino­
cycline was applied in the primary microglial cells. Treatment of
microglial cells with gabapentin (100 µM) for 2 h increased the gene
expression of IL-10 and POMC, whereas pretreatment of minocycline
(60 µM) for 1 h did not affect the baseline of either gene expression.
However, minocycline entirely inhibited gabapentin-stimulated IL-10
and POMC gene expression (Fig. 8A-8B). Likewise, minocycline blocked
gabapentin-increased the protein levels of IL-10 and β-endorphin
(Fig. 8C-8D).
3.4. Intrathecal microglial inhibitor, Il-10 antibody and β-endorphin
antiserum and μ-opioid receptor (MOR) antagonist suppressed
gabapentinoids-inhibited mechanical allodynia
To test the fundamental role of spinal microglial expression of IL-10
and β-endorphin in gabapentinoids alleviated mechanical allodynia, the
microglial inhibitor minocycline was first applied. Two groups of
neuropathic rats were firstly injected intrathecally with saline (10 μL) or
minocycline (100 μg), followed by second injection intrathecally of
gabapentin (100 μg), pregabalin (30 μg) or mirogabalin (10 μg) 4 h later.
The dose regimens used in minocycline treatment were from previous
reports (Mei et al., 2011; Tang et al., 2020; Shoaib et al., 2019). The
hindpaw withdrawal thresholds to mechanical stimuli were evaluated
before and 0.5, 1, 2, and 4 h after gabapentinoids treatment. As shown in
Fig. 9A-9C, intrathecal gabapentin, pregabalin and mirogabalin time-
dependently inhibited mechanical allodynia. Minocycline did not
significantly alter the baseline withdrawal thresholds in the contralat­
eral or ipsilateral hindpaws, but entirely blocked gabapentinoids-
inhibited allodynia.
In addition, two groups of neuropathy rats were firstly injected
intrathecally with saline (10 μL) or the IL-10 antibody (2 μg), followed
by second injection intrathecally of gabapentin (100 μg), pregabalin (30
μg) or mirogabalin (10 μg) 0.5 h later. Intrathecal gabapentinoids time-
dependently inhibited mechanical allodynia. Although it did not alter

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Fig. 5. Effects of intrathecal gabapentin on mechanical allodynia and thermal hyperalgesia (A, B), IL-10 and POMC mRNA expression (C, D), IL-10 and β-endorphin
expression (E, F), co-labeling of IL-10 with the microglial marker Iba-1 in spinal dorsal horns laminae I-V (scale bar: 50 µm) and the % immunolabeled surface areas
of IL-10/Iba-1 (G, H) in male sham rats and male and female neuropathic rats. The data are presented as means ± SEM (n = 6 per group). * P < 0.05 compared with
saline group, by unpaired and two-tailed Student t-test.

the baseline withdrawal responses in the contralateral or ipsilateral
hindpaws, IL-10 antibody completely prevented gabapentinoids from
countering mechanical allodynia (Fig. 9D-9F).
Furthermore, two groups of neuropathy rats were injected firstly
with the blank serum (1:10, 10 μL) or the β-endorphin antiserum (1:10,
10 μL) intrathecally, followed by the another injection of gabapentin
(100 μg), pregabalin (30 μg) or mirogabalin (10 μg) intrathecally 0.5 h
later. The intrathecal pretreatment with the β-endorphin antiserum did
not affect the control withdrawal responses in the contralateral or ipsi­
lateral hindpaws, but considerably attenuated gabapentinoids-inhibited
mechanical allodynia (Fig. 10A-10C).
Lastly, four groups of neuropathy rats injected firstly with normal
saline (10 µL), the MOR antagonist CTAP (10 µg), κ-opioid receptor
(KOR) antagonist GNTI (50 µg) or δ-opioid receptor (DOR) antagonist
naltrindole (5 µg) intrathecally, followed by another injection of gaba­
pentin (100 µg), pregabalin (30 µg) or mirogabalin (10 µg) intrathecally
0.5 h later. The doses and regimen of CTAP, GNTI and naltrindole were
based on the previous publication (Steinmiller and Young, 2008). As
shown in Fig. 10D-10F, intrathecal pretreatment with CTAP did not have
significant effect on the response of withdrawal in the contralateral or
ipsilateral hind paws, but nearly wholly attenuated spinal
gabapentinoids-inhibited mechanical allodynia. In contrast, GNTI or
naltrindole failed to block gabapentinoids-inhibited mechanical
allodynia.

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Fig. 6. Effects of gabapentin, pregabalin and mirogabalin on IL-10 (A-C) and POMC (D-F) mRNA expression in microglial, astrocytic and neuronal cells. G-H
Concentration-response curves of gabapentinoids on IL-10 and POMC mRNA expression. Cells were incubated with gabapentinoids for 2 h and mRNA expression of
IL-10 and POMC were evaluated by using real-time qPCR. The data are presented as means ± SEM (n = 6 per group). * P < 0.05 compared with control group, by one-
way ANOVA followed by the post-hoc Bonferroni test.
3.5. No expression of the α2δ-1 subunit of the voltage-gated calcium
channel in microglia
To figure out the target molecule responsible for gabapentinoids-
induced microglial IL-10 and β-endorphin expression, specific cellular
expression of the α2δ-1 subunit of voltage-gated calcium channel was
first assessed in the ipsilateral spinal cords frozen sections, using co-
labeling immunofluorescence of α2δ-1 with NeuN, GFAP or Iba-1 anti­
bodies. Two groups of neuropathic rats received an intrathecal injection
of normal saline (10 μL) or gabapentin (100 μg) and the spinal lumbar
enlargements were obtained 1 h after injection. The α2δ-1 immunoflu­
orescence was co-localized with NeuN in the spinal dorsal horn
(Fig. 11A, 11B), but not with GFAP (Fig. 11C, 11D) or Iba-1 (Fig. 11E,
11F). Intrathecal injection of gabapentin did not alter the immuno­
staining of α2δ-1 with NeuN (Fig. 11G, 11H), GFAP (Fig. 11I, 11 J) or
Iba-1 (Fig. 11K, 11L), which was also confirmed by quantitative mea­
surement of α2δ-1/NeuN (Fig. 11M), α2δ-1/GFAP (Fig. 11N) or α2δ-1/
Iba-1 (Fig. 11O) using the ImageJ Software. Additionally, western blot
analysis also confirmed the expression of α2δ-1 in the spinal homoge­
nates of neuropathic rats and intrathecal gabapentin (100 µg) did not
significantly influence α2δ-1 protein expression compared to saline
control (Fig. 11P).
Likewise, double immunofluorescence staining was performed in
primary cultured spinal cells. Primarily cultured cells were incubated
with the vehicle or gabapentin (100 µM) for 2 h. As shown, α2δ-1 was co-
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Brain Behavior and Immunity 95 (2021) 344–361
localized with NeuN-positive neurons (Fig. 12A, 12B). However, α2δ-1
was not expressed in GFAP-positive astrocytes (Fig. 12C, 12D) or Iba-1-
positive microglial cells (Fig. 12E, 12F). Moreover, treatment with
gabapentin for 2 h did not affect the expression of α2δ-1 in neurons
(Fig. 12G, 12H), astrocytes (Fig. 12I, 12 J) or microglial cells (Fig. 12K,
12L). Furthermore, quantitative measurement confirmed that α2δ-1 was
expressed in neurons but not astrocytes or microglial cells, and gaba­
pentin treatment did not alter the α2δ-1 expression either in neurons,
astrocytes or microglial cells (Fig. 12M-12O).
Finally, to test whether gabapentinoids-inhibited allodynia was
through activation of the spinal GLP-1Rs, GPR40, CRF receptors or
adenosine A1 receptors, their specific antagonists were applied. The
doses and regimen of the GLP-1R antagonist exendin9-39 (Göke et al.
1993), GPR40 antagonist GW1100 (Nakamoto et al. 2012), CRF receptor
antagonist α-helical CRF(9–41) (Sawamura et al. 2003) and adenosine
A1 receptor antagonist DPCPX (Park and Jun, 2008) were based on the
previous papers. Twelve groups of neuropathy rats injected firstly saline
(10 µL), exendin9-39 (2 µg), GW1100 (300 µg) and α-helical CRF9-41
(20 µg) intrathecally, followed by another injection of gabapentin
(100 µg), pregabalin (30 µg) or mirogabalin (10 µg) 0.5 h later. In
comparison with the control, intrathecal pretreatment with exendin9-
39, GW1100 or α-helical CRF9-41 did not significantly reduce the
inhibitory effects of gabapentin (Fig. 13A), pregabalin (Fig. 13B) or
mirogabalin (Fig. 13C) on mechanical allodynia. In addition, six groups
of neuropathy rats injected firstly saline (10 µL) or DPCPX (10 µg)
K.A. Ahmad et al.
Fig. 7. Effects of gabapentin, pregabalin and mirogabalin on IL-10 (A-C) and β-endorphin (D-F) protein expression in microglial, astrocytic and neuronal primary
culture cells. The cultured medium was obtained 2 h after gabapentinoids incubation. The data are presented as means ± SEM (n = 6 per group). * P < 0.05 compared
with the control group, by one-way ANOVA followed by the post-hoc Bonferroni test.
intrathecally, followed by another injection of gabapentin (100 µg),
pregabalin (30 µg) or mirogabalin (10 µg) 0.5 h later. As shown in
Fig. 13D-13F, intrathecal prior-treatment with DPCPX failed to inhibit
gabapentinoids-inhibited mechanical allodynia.
4. Discussion
Gabapentinoids have been extensively tested in various animal
models of nociceptive pain and pain hypersensitivity, particularly in
neuropathic pain (Cheng and Chiou 2006). However, there have been
few reports that compared their dose–response curves head to head. Our
results demonstrated that a single intrathecal injection of each gaba­
pentinoid quickly inhibited mechanical allodynia and thermal hyper­
algesia in the ipsilateral hind paws of neuropathic rats. Gabapentinoids
exhibited time-dependent inhibition of allodynia, with peak 1 h after
intrathecal administration. Their dose-dependent inhibitions of me­
chanical allodynia were projected with Emax values 66%, 61% and 65%
MPE, and ED50 values 30.3 µg (176.9 nmol), 6.2 µg (38.9 nmol) and 1.5
µg (7.2 nmol) for gabapentin, pregabalin and mirogabalin respectively.
The results indicate that mirogabalin is the most potent compound with
the ED50 value being 4.4- and 23.6-fold less than that of pregabalin and
gabapentin respectively, in agreement with the previous findings that
mirogabalin produced more potent analgesic effects than pregabalin in
the rat models of peripheral diabetic neuropathy and spinal cord injury
(Domon et al., 2018a, 2018b; Domon, Kitano, and Makino 2018) and
pregabalin exhibited higher potency analgesia than gabapentin in a
clinical trial (Bockbrader et al. 2010).
Remarkably, our study illustrated that gabapentinoids alleviate
mechanical allodynia through de novo IL-10 and subsequent β-endor­
phin microglial expression in neuropathic pain. Microglia are resident
macrophages in the spinal cord and involved pain process. It has been
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Brain Behavior and Immunity 95 (2021) 344–361
shown that microglia release antiinflammatory cytokines such as IL-10,
IL-4, NGF, GDNF, and opioid peptides such as β-endorphin and dynor­
phin A, which inhibit sensory transmission and central sensitization
leading to pain relieve in neuropathic pain (Wu et al., 2017a, 2017b,
2018a; Gong et al., 2014; Franco and Fernandez-Suarez, 2015; Carniglia
et al., 2017). IL-10 is considered as the most prominent antiin­
flammatory and immunosuppressive cytokine so far, through binding to
IL-10 receptor-α (Sabat et al., 2010; Glocker et al., 2011). IL-10 displays
marked antinociceptive activities in spinal nerve pain, chronic
constriction injury, chemotherapy, peripheral diabetic pain, bone can­
cer pain and CFA-induced inflammatory pain (Wu et al., 2018a; Milligan
et al., 2005; Ledeboer et al., 2007; Zhou et al., 2008; Kim et al., 2011;
Thakur et al., 2016). It has been documented that IL-10 relieves nerve
pain via promotion of microglial β-endorphin expression (Wu et al.
2018a).
Our current data showed that treatment with gabapentin, pregabalin
and mirogabalin for 2 h specifically stimulated IL-10 mRNA and protein
expression of microglial cells. Their stimulation on IL-10 expression was
not observed in astrocytic or neuronal cells. It is known that IL-10
treatment or gene transfer stimulated microglial β-endorphin expres­
sion and not vice versa (Wu et al. 2017b), or upregulated POMC in
arcuate nucleus in hypothalamus in leptin-substituted rats (Nakata et al.
2016). Indeed, treatment of microglial cells with gabapentinoids spe­
cifically stimulated expression of β-endorphin but not dynorphin A.
Additionally, concentration–response analysis showed the EC50 values
for gabapentin, pregabalin and mirogabalin to promote IL-10 gene were
41.3, 11.5 and 2.5 µM respectively, which were overlapped with their
EC50 values 34.7, 13.3 and 2.8 µM of POMC gene upregulation. These
overlapped EC50 values were much greater than their α2δ-1 binding
affinities in nanomolar range (Domon et al., 2018a, 2018b; Li et al.,
2011; Suman-Chauhan et al., 1993) but close to those of physiological
K.A. Ahmad et al. Brain Behavior and Immunity 95 (2021) 344–361

Fig. 8. Inhibitory effect of the microglial

inhibitor minocycline on gabapentin-
stimulated gene (A, B) and protein expres­
sion (C, D) of IL-10 and β-endorphin in pri­
mary cultures of microglia. Minocycline was
applied 1 h before gabapentin treatment,
and microglial cells and the cultured me­
dium were obtained 2 h later. The data are
presented as means ± SEM (n = 6 per
group). *, # P < 0.05 compared with the
control or gabapentin groups respectively,
by one-way ANOVA followed by the post-
hoc Bonferroni test.

Fig. 9. Inhibitory effects of intrathecally microglial inhibitor minocycline (A-C) and specific IL-10 antibody (D-F) on spinal gabapentinoids-inhibited mechanical
allodynia in neuropathic rats. Minocycline was given 4 h before gabapentinoids treatment, while the IL-10 antibody was injected 0.5 h prior to gabapentinoids
treatment. The data are shown as means ± SEM (n = 6 per group). * P < 0.05 vs. the saline + gabapentin, pregabalin or mirogabalin group, by repeated measures
two-way ANOVA followed by the post-hoc Bonferroni test.

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K.A. Ahmad et al.
Fig. 10. Blockage effects of intrathecally β-endorphin antiserum (A-C) and opioid receptor subtype antagonists (D-F) on spinal gabapentinoids-inhibited mechanical
allodynia in neuropathic rats. The β-endorphin antiserum and opioid receptor antagonists CTAP, GNTI, and naltrindole were administrated intrathecally 0.5 h before
spinal gabapentinoids administration. The data are presented as means ± SEM (n = 6 per group). * P < 0.05 vs. the saline + gabapentin, pregabalin or mirogabalin
group, by repeated multiple measures two-way ANOVA followed by the post-hoc Bonferroni test.
actions, which are usually in the low micromolar range (Stahl et al.
2013). Further, the EC50 values of these three gabapentinoids on IL-10 or
POMC gene expression were significantly correlated to their ED50 values
on mechanical allodynia (r = 0.999 or 0.988, n = 3 each, P = 0.002 or
0.024). The positive correlation for gabapentinoids to stimulate IL-10/
β-endorphin and to inhibit mechanical allodynia was further supported
by their spinal stimulation with different doses. Intrathecal injection of
gabapentin (100 µg), pregabalin (30 µg) and mirogabalin (10 µg), by the
same degrees, stimulated fast gene and protein expression (within 1 h of
injection) of β-endorphin and IL-10 (but not dynorphin A) in spinal
neuropathy rats, which was parallel to their time-course of allodynia.
Co-labeling immunostaining further indicated that gabapentin upregu­
lated β-endorphin and IL-10 in spinal microglial, but was not observed in
astrocytic or neuronal cells. Our outcomes are in harmony with previous
reports that intrathecal gabapentin significantly increased spinal IL-10
expression and subsequently decreased proinflammatory cytokines
expression in neuropathic rats and morphine tolerant rats (Lee et al.,
2013; Bao et al., 2014b, 2014a).
Microglia activated by nerve injury, trauma or lipopolysaccharide
(LPS) treatment react quickly by promoting the classical proin­
flammatory system (M1 phenotype). M1 polarized microglia secret large
amounts of inflammatory factors implicated in the progress of neuro­
pathic pain (Milligan and Watkins, 2009; Perry and Holmes, 2014).
Reactive microglia undergo transformations in phenotype, including
activation of the p38-MAPK (probably p38α-MAPK subtype) signaling
pathway in neuropathic pain, which can be inhibited by minocycline
(Hua et al., 2005; Wu et al., 2017a, 2017b, 2018a). In contrast, microglia
can develop an alternative beneficial system (M2 phenotype), charac­
terized by the expression of the anti-inflammatory and neuroprotective
factors such as IL-10, TGF-β, IL-4, and endogenous opioids peptides
(Franco and Fernandez-Suarez, 2015; Wu et al., 2017a, 2017b, 2018b;
Huang et al., 2016; Li et al., 2016). Expression of the anti-inflammatory
and neuroprotective factors may not require microglial activation
(p38α-MAPK), but needs p38β-MAPK activation which can also be
inhibited by minocycline (Wu et al., 2017a, 2017b, 2018b; Li et al.,
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Brain Behavior and Immunity 95 (2021) 344–361
2017; Huang et al., 2017). Remarkably, gabapentinoids stimulated IL-10
and β-endorphin expression in primary microglial cells in resting states
(without LPS stimulation). Also, pretreatment with minocycline entirely
inhibited gabapentin-stimulated IL-10 and β-endorphin expression in
primary cultures of microglia. Furthermore, intrathecal gabapentin
induced expression of spinal IL-10 and β-endorphin in sham and
neuropathic rats by nearly the same degrees. However, spinal gaba­
pentin did not significantly affect nociceptive behaviors in sham rats,
conceivably due to lower peripheral neuronal sensitivity to endogenous
opioid peptides in normal nociceptive circumstances in which central
sensitization is not present (Gong et al. 2014). This observation is
consistent with previous findings where GLP-1 receptor agonists,
α7nAChR agonists, GPR40 agonists and electroacupuncture induced
microglial IL-10 and β-endorphin expression and aconitines stimulated
dynorphin A expression by almost same degrees in primary microglial
cells in the presence and absence of LPS, and in both contralateral and
ipsilateral spinal cords of neuropathic rats (Mao et al., 2019; Apryani
et al., 2020; Gong et al., 2014; Ali et al., 2020; Fan et al., 2015; Huang
et al., 2016; Li et al., 2016). In addition, sexual dimorphism has been
suggested in nociceptive role of microglia, where nerve injury-induced
spinal proinflammatory cytokine expression and mechanical allodynia
hypersensitivity were involved only in male animals (Sorge et al. 2015).
However, intrathecal gabapentin produced nearly similar pain anti­
hypersensitivity and spinal microglial IL-10 and β-endorphin expression
irrespective of the sex. Our findings are in agreement with previous
studies where no sexual dimorphism is found in microglia-related anti­
hypersensitive effects on peripheral neuropathic pain, e.g., IL-10 (Wu
et al. 2018a), α7nAChR agonists (Apryani et al. 2020) and electro­
acupuncture (Ali et al. 2020), or genetic deficiency of the microglial
selective molecules including P2Y12 (Gu et al. 2016), CX3CR1 (Stani­
land et al. 2010) and TMEN16F (Batti et al. 2016). Furthermore, various
female chronic pain models have shown pain inhibitory effects of
microglial TLR4 knockdown (Lan et al. 2010), CX3CR1 antibody (Hu
et al. 2012), cathepsin S inhibitor (Nieto et al. 2016), and P2X7 antag­
onists (Yang et al., 2015; Nieto et al., 2016) similar to males. Our results
K.A. Ahmad et al. Brain Behavior and Immunity 95 (2021) 344–361

Fig. 11. Effects of gabapentin (100 µg) administered intrathecally on spinal α2δ-1 expression in neuropathic rats. Co-labeling immunostaining of α2δ-1 with the
neuronal (NeuN) marker, astrocytic (GFAP) marker or microglial (Iba-1) marker in the spinal dorsal horn (scale bar: 250 µm) and the I-V laminate (scale bar: 50 µm,
A-F). Yellow co-labeling of α2δ-1 and cellular markers shown by arrows. G-L. Effect of gabapentin (100 µg), given intrathecally for 1 h, on α2δ-1 expression in
neurons, astrocytes or microglial cells. The % immunolabeled surface areas of α2δ-1/NeuN (M), α2δ-1/GFAP (N), and α2δ-1/Iba-1 (O) from spinal dorsal horn
laminae I-V were determined by the ImageJ software. Western blot of α2δ-1 protein expression (P). Data are shown as means ± SEM (n = 5–6 per group). (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

also support the clinical practice that gabapentinoids do not exhibit
gender deference in anti-neuropathic pain efficacies.
Given the existed correlation between IL and 10/β-endorphin
microglial stimulation and mechanical allodynia inhibition, the causal
relationship of gabapentinoids was further explored. Minocycline is an
effective microglial activation inhibitor, although it may have minor
effects on astrocytes or neurons (Yoon et al., 2012; Jackson-Lewis et al.,
2002; Jin et al., 2014). It has been accepted that minocycline may
prevent the initiation or early development of neuropathy pain, but does
not produce antinociception in established neuropathy (Raghavendra
et al., 2003; Li et al., 2016; Gong et al., 2014; Mei et al., 2011; Fan et al.,
2016). In addition, minocycline has been successfully proved to block
microglia-mediated antinociceptive effects of GLP-1R agonist exenatide,
GPR40 agonist GW9508, α7nAChR agonist cinobufagin, aconitum-
derived bullatine A, aconitine, lappaconitine and bulleyaconitine A but
not neurons-mediated antinociceptive effects of MOR agonists morphine
and β-endorphin, KOR agonist dynorphin A, and glycine receptor
agonists glycine, gelsemine and koumine (Huang et al., 2016; Shoaib
et al., 2019; Li et al., 2016; Gong et al., 2014; Apryani et al., 2020; Mao
et al., 2019; Fan et al., 2016). In our current study, intrathecal pre­
treatment with minocycline completely blocked gabapentinoids-
inhibited mechanical allodynia. Additionally, intrathecal pretreatment
with the IL-10 antibody totally attenuated each gabapentinoid
compound-inhibited mechanical allodynia, which are supported by that
intrathecal IL-10 antibody blocked gabapentin antinociception in
neuropathic rats and morphine tolerant rats (Lee et al., 2013; Bao et al.,
2014b, 2014a). Furthermore, prior-treatment intrathecally with
β-endorphin antiserum and MOR antagonist, but not KOR or DOR an­
tagonists, entirely alleviated spinal gabapentinoids-inhibited mechani­
cal allodynia, suggesting a subsequent role of endogenous opioid role in
gabapentinoids analgesia, in agreement with that the opioid-receptor
antagonist naloxone alleviated gabapentin-induced antinociception in
the rat orofacial formalin test (Miranda et al. 2015).
The importance of the microglial IL-10/β-endorphin pain

356
K.A. Ahmad et al. Brain Behavior and Immunity 95 (2021) 344–361

Fig. 12. Representative photomicrographs of the specific α2δ-1 expression in primarily cultured spinal neuronal cells (A, B), but not astrocytic cells (C, D) or
microglial cells (E, F). DAPI staining was used to identify cell nuclei. G-L. Effects of gabapentin treatment (100 µM) for 2 h on α2δ-1 expression in neurons, astrocytes
or microglial cells. The % immunolabeled surface areas of α2δ-1/NeuN (M), α2δ-1/GFAP (N), and α2δ-1/Iba-1 (O) were determined by the ImageJ software. Scale
bars: A, C, E, G, I, K, 50 µm; B, D, F, H, J, L, 10 µm. Data are shown as means ± SEM (n = 5–6 per group).

Fig. 13. Effects of the intrathecally GLP-1 receptor antagonist exendin-9–39, GPR40 antagonist GW1100, CRF receptor antagonist α-helical CRF (9–41) (A-C) and
adenosine A1 receptor antagonist DPCPX (D-F) on spinal gabapentinoids-inhibited mechanical allodynia in neuropathic rats. Exendin9-39, GW1100, α-helical CRF9-
41 or DPCPX was injected intrathecally 0.5 h before spinal gabapentinoids administration. The data are presented as means ± SEM (n = 6 per group).

357
K.A. Ahmad et al.
Fig. 14. Schematic of the proposed mechanisms of gabapentinoids to inhibit pain hypersensitivity in neuropathic rats following activation of microglial spinal IL-10/
β-endorphin signaling pathway. Gabapentinoids stimulate IL-10, which subsequently expresses β-endorphin following activation of IL-10 receptors on microglial cells
by an autocrine signaling. β-Endorphin passes the microglial-neuronal synapse to activate μ-opioid receptor (MOR) in neurons and produce antinociception.
antihypersensitivity signal transduction has been selectively addressed.
The GLP-1R agonist exenatide and morroniside, GPR40 agonist
GW9508, α7nAChRs agonists PHA-543613, cinobufagin and lemairamin
inhibited mechanical allodynia and thermal hyperalgesia in various pain
models through spinal microglial expression of IL-10 and subsequent
β-endorphin expression (Gong et al. 2014; Wu et al. 2017; Mao et al.,
2019; Apryani et al., 2020; Wang et al., 2020; Tang et al., 2020). Low
frequency electroacupuncture stimulation produced pain anti­
hypersensitivity by spinal microglial IL-10/β-endorphin upregulation
(Ali et al. 2020), probably by activation of α7nAChR (Wang et al. 2018).
Early life-induced constitutive suppression of neuropathic pain is
mediated by the IL-10/β-endorphin microglial signaling pathway
(McKelvey et al., 2015; Tang Xue-qi et al., 2019). Thus the discovery of
mechanism of gabapentinoids analgesia expands biological implications
of the IL-10/β-endorphin spinal microglial pain antihypersensitivity
pathway. In addition, the pathway might be a promising human-
validated target for discovery and development of analgesics in neuro­
pathic pain and other pain hypersensitivity states, as gabapentinoids are
clinically effective analgesics. Furthermore, the effects of gabapenti­
noids may characterize microglia-mediated analgesics. Our results
showed that gabapentinoids specifically attenuated ipsilateral mechan­
ical allodynia without influencing the contralateral paws thresholds
significantly. The results are in concurrence with previous reports in
which gabapentinoids were efficient in attenuating pain hypersensitiv­
ity in models of neuropathy, bone-cancer, inflammatory and post­
operative pain, but not nociceptive pain such as hot-plate, tail-
immersion or paw-pressure test (Cheng and Chiou 2006). In addition, all
of gabapentinoids exhibit the same efficacy of around 60–70% MPE in
attenuating mechanical allodynia, which is much lower than 100% MPE
or greater (changing thresholds in normal nociceptive pain states) pro­
duced by morphine or other opioid analgesics (Wang et al., 2017; Mao
et al., 2020). All these results provide the basis for gabapentinoids
clinical indications of postherpetic neuralgia, peripheral diabetic neu­
ropathy and fibromyalgia, as well as postoperative pain, headache,
chronic low back pain and visceral pain, but not nociceptive pain, and
probably define their relatively limited clinical efficacy. Indeed, our
extensive dose–response studies revealed that microglia-mediated anti­
nociceptive effects, produced by agonists of IL-10 receptors, GLP-1Rs,
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Brain Behavior and Immunity 95 (2021) 344–361
GPR40 and α7nAChRs, cynandione A, and aconitines, are specific in
pain hypersensitivity (not nociceptive pain) with efficacies of around
60–70% MPE in mechanical allodynia (Gong et al., 2014; Wu et al.,
2017b, 2018a; Li et al., 2016; Huang et al., 2016, 2017; Apryani et al.,
2020; Sun et al., 2018).
The target molecules for gabapentinoids to fast induce IL-10/
β-endorphin microglial expression have not been illustrated. It has been
reported gabapentinoids did not displace radioligands from widely-
studied target sites, such as adrenergic, adenosine, dopamine, seroto­
nin, histamine, GABA, acetylcholine, glutamate and opioid receptors,
calcium and potassium channels, and dopamine, norepinephrine and
serotonin transmitters up to 50 or 100 μM (Domon et al., 2018a, 2018b;
Li et al., 2011). In addition, GLP-1Rs and GRP40 are expressed in
microglia and their agonists stimulated microglial IL-10/β-endorphin
expression (Mao et al., 2019; Wu et al., 2017a, 2017b). Alternatively,
CRF receptors have been associated with the microglial β-endorphin
expression (Iwaszkiewicz et al. 2013) and adenosine A1 receptors
antagonism has been shown to prevent gabapentin behavioral effects
(Martins et al. 2015). However, our results demonstrated that the an­
tagonists of GLP-1Rs, GPR40, CRF receptors and adenosine A1 receptors
were unable to block each gabapentinoid-inhibited mechanical allody­
nia, suggesting that their spinal IL-10/β-endorphin expression is not
mediated by these receptors. Moreover, specific colocalization of α2δ-1
was observed with the neuronal biomarker, but not with microglial or
astrocytic biomarker in the spinal dorsal horns from neuropathy rats and
primary cultures of spinal cells originated from neonatal rats. In addi­
tion, gabapentin treatment for 2 h did not affect α2δ-1 expression either
in neurons or even in microglia and astrocytes. Our findings are in
agreement with the previous studies that α2δ-1 was expressed only in
neurons but not microglia or astrocytes (Yang et al. 2012). Thus, our
results reveal that gabapentinoids-stimulated microglial IL-10/
β-endorphin expression was not through α2δ-1 binding or interaction.
Consequently, additional researches are warranted to search for the
target molecules of gabapentinoids.
To sum up, intrathecal gabapentinoids quickly inhibited mechanical
allodynia and thermal hyperalgesia in neuropathic rats. They also pro­
moted IL-10 and β-endorphin expression in spinal dorsal horn microglia
and primary microglia cultures, which were absent in α2δ-1 expression.
K.A. Ahmad et al.
In addition, gabapentinoids-inhibited allodynia was entirely reversed by
prior-treatment with microglial metabolic inhibitor minocycline, IL-10
antibody, β-endorphin antiserum, and MOR antagonist CTAP. The re­
sults suggest that spinal gabapentinoids inhibit neuropathic pain
through microglial expression of IL-10 and subsequent β-endorphin
expression. The proposed schematic microglial pathway of IL-10/
β-endorphin for gabapentinoids to alleviate neuropathic pain is shown
in Fig. 14.
Funding sources
This study was supported by the National Natural Science Founda­
tion of China (No. 81673403).
Author contributions
Conceived and designed the experiments: KAA and YXW; performed
the experiments: KAA, RMS, MZA, MD, ML, EA and XYL; analyzed the
data: KAA and YXW and wrote the paper: KAA and YXW.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
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