International Immunopharmacology 106 (2022) 108622

Contents lists available at ScienceDirect

International Immunopharmacology
journal homepage: www.elsevier.com/locate/intimp

Inhibition of pan-Aurora kinase attenuates evoked and ongoing pain in

nerve injured rats via regulating KIF17-NR2B mediated signaling
Ankit Uniyal a, Akhilesh a, Aaina Singh Rathore b, Priyanka Kumari Keshri b,
Surya Pratap Singh b, Sanjay Singh c, Vinod Tiwari a, *
a
Neuroscience and Pain Research Laboratory, Department of Pharmaceutical Engineering and Technology, Indian Institute of Technology (Banaras Hindu University),
Varanasi 221005, Uttar Pradesh, India
b
Department of Biochemistry, Institute of Science, Banaras Hindu University, Varanasi, 221005, India
c
Baba Saheb Bhim Rao Ambedkar Central University (BBAU), Lucknow 226025, Uttar Pradesh, India

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

Keywords: Kinesins (KIF’s) are the motor proteins which are recently reported to be involved in the trafficking of noci­
Aurora Kinase ceptors leading to chronic pain. Aurora kinases are known to be involved in the regulation of KIF proteins which
Kinesin are associated with the activation of N-methyl-D-aspartate (NMDA) receptors. Here, we investigated the effect of
Spontaneous ongoing pain
tozasertib, a pan-Aurora kinase inhibitor, on nerve injury-induced evoked and chronic ongoing pain in rats and
NR2B
NMDA
the involvement of kinesin family member 17 (KIF17) and NMDA receptor subtype 2B (NR2B) crosstalk in the
Neuroinflammation same. Rats with chronic constriction injury showed a significantly decreased pain threshold in a battery of pain
behavioural assays. We found that tozasertib [10, 20, and 40 mg/kg intraperitoneally (i.p.)] treatment showed a
significant and dose-dependent inhibition of both evoked and chronic ongoing pain in rats with nerve injury.
Tozasertib (40 mg/kg i.p.) and gabapentin (30 mg/kg i.p.) treatment significantly inhibits spontaneous ongoing
pain in nerve injured rats but did not produce any place preference behaviour in healthy naïve rats pointing
towards their non-addictive analgesic potential. Moreover, tozasertib (10, 20, and 40 mg/kg i.p.) and gabapentin
(30 mg/kg i.p.) treatment did not altered the normal pain threshold in healthy naïve rats and didn’t produce
central nervous system associated side effects as well. Western blotting and reverse transcription polymerase
chain reaction studies suggested enhanced expressions of NR2B and KIF-17 along with increased nuclear factor
kappa β (NFkβ), tumour necrosis factor-α (TNF-α), interleukin 1β (IL-1β), and interleukin 6 (IL-6) levels in dorsal
root ganglion (DRG) and spinal cord of nerve injured rats which was significantly attenuated on treatment with
different does of Tozasertib. Findings from the current study suggests that inhibition of pan-Aurora kinase
decreased KIF-17 mediated NR2B activation which further leads to significant inhibition of evoked and chronic
ongoing pain in nerve-injured rats.

1. Introduction effects including but not limited to sedation, motor incoordination, drug
addiction and development of tolerance [4]. The pathophysiology of
Chronic pain is a devastating disorder affecting more than 20% of the neuropathic pain has both nociceptive and inflammatory components
population worldwide [1,2]. A subtype of this is chronic neuropathic and requires an effective strategy to mitigate the same. N-methyl-
pain which occurs due to the damage to the somatosensory nervous aspartate-receptors (NMDAR’s) are known to be involved in the devel­
system including peripheral nerve fibers such as Aδ, Aβ, and C fibres. opment and maintenance of neuropathic pain both in preclinical and
The prevalence of neuropathic pain ranges from 6 to 10 % globally [3] clinical studies [5,6]. A subset of this receptor unit, NMDA receptor
and the patients suffering from the same exhibits significant hyper­ subtype 2B (NR2B) is essential for the localization of NMDARs into the
algesia, shooting sensations, numbness, allodynia, and spontaneous synaptic membrane [7] and plays an important role in learning and
ongoing pain. Unfortunately, the currently available analgesics provide memory and chronic pain. Central sensitization which is a primary
only mild symptomatic relief and that too at the cost of several side feature of neuropathic pain pathophysiology develops due to the over-

* Corresponding author at: Department of Pharmaceutical Engineering & Technology, Indian Institute of Technology (B.H.U.), Varanasi 221005, U.P., India.
E-mail address: [email protected] (V. Tiwari).

https://doi.org/10.1016/j.intimp.2022.108622
Received 24 November 2021; Received in revised form 28 January 2022; Accepted 10 February 2022
Available online 17 February 2022
1567-5769/© 2022 Elsevier B.V. All rights reserved.
A. Uniyal et al. International Immunopharmacology 106 (2022) 108622

activity of NMDARs at the spinal and supra-spinal regions [8,9]. with standard laboratory food ad-libitum and sterile water. All the ani­
Kinesins are the motor proteins which are involved in the trafficking mals were randomly assigned to the different experimental groups (n =
of proteins in different cell types including neurones. In highly polarised 8/group).
neurons the delivery and sorting of organelle is also carried out with the
help of kinesin superfamily (KIFs). Any impairment in the expression 2.3. Ethical Committee approval
and functioning of KIFs results in maladaptive neuronal circuits that
results in improper propagation of neuronal signals [10,11]. Kinesin All experiments were performed in accordance with the guidelines
family member 17 (KIF17) is a homodimeric kinesin motor protein that by International Association for The Study of Pain and the Committee
belongs to the osmotic avoidance abnormal protein 3 (OSM3)/KIF17 for the Purpose of Control and Supervision of Experiments on Animals
family. These adenosine tri-phosphate (ATP)-dependent motors trans­ (CPCSEA), Government of India, New Delhi. All the experimental pro­
port the synthesized cargos in a retrograde direction to the +end of tocols were approved by the Institute Animal Ethics Committee of
synaptic membrane. KIF17 consists of the tail domain that binds to the Banaras Hindu University, Varanasi, UP, India (Dean/2019/IAEC/
postsynaptic density-95/Discs large/Zona occludens-1 (PDZ) domain of 1617).
mammalian lin ten protein (mLin-10). Further, the mLin 10 binds to the
large scaffolding cargo complex that includes the NR2B subunit of
2.4. Animal model and experimental design
NMDAR [4]. The dynamic living mammalian neuron studies have
confirmed the trafficking of NR2B subunit by KIF17 kinesin nanomotors
Chronic constriction injury in the left sciatic nerve of the rats was
[12].
performed for the development of neuropathic pain as per the method
Regardless of demonstrated preclinical effectiveness, the NMDA re­
described earlier [15,21,22]. Rats were briefly anesthetized using ke­
ceptor antagonist failed to treat the neuropathic pain in clinical trials
tamine [90 mg/kg intraperitoneally (i.p.)] and xylazine (10 mg/kg i.p.).
[13,14]. The major reason behind this is NMDA receptor interaction
Under aseptic conditions, the left sciatic nerve of the rats was exposed
with other nociceptive units and secondary messenger systems. Another
through bicep femoris muscle. The nerve was then made free from the
issue with the use of NMDA antagonists is the occurrence of severe
surrounding tissue and ligated with a 6–0 braided silk suture (Teleflex
adverse effects which further suppress the clinical utility of this class of
Medical, USA). Each ligation was confirmed with the very short reflex of
pharmacological agents [14]. However, because of significant involve­
the ipsilateral paw. After the ligation of the sciatic nerve, the muscles
ment of NMDA receptor system during chronic neuropathic pain its
followed by skin were sutured using 4–0 silk threads. Finally, povidone-
therapeutic targeting cannot be ignored. Therefore, an indirect
iodine solution 10% w/v was applied to the sutured skin of rats. Animals
approach of targeting the NMDA receptor function by interfering with
were closely monitored until they recovered from anaesthesia followed
receptor maturation, synthesis, and transport to the synaptic membrane
by placing them in their home cages.
could be an effective strategy for the treatment of neuropathic pain.
For pain behaviour and molecular biology studies rats were divided
Previous findings from our lab suggests that inhibition of pan-Aurora
in to six experimental groups with minimum eight rats/group. First
kinase regulates the expression of KIF11 which is a regulator of shape
group consists of naïve healthy rats, while second group was disease
of dendrites in matured neurons [15]. Importantly, KIF17 has been
control group where nerve-injured rats were administered with vehicle
recently known to be involved in pathophysiology of chronic pain
of tozasertib. Rats in third, fourth and fifth group belonged to different
[16–18]. Thus, exploring the exogenous ligands that interfere with the
test groups where nerve injured rats were treated with intraperitoneal
KIF17-NR2B cargo system can provide a novel line of therapeutics for
tozasertib at 10 mg/kg, 20 mg/kg and 40 mg/kg respectively. Sixth
the management of neuropathic pain. Aurora kinase are the enzymes
group consists of the standard control group in which nerve injured rats
that regulates the molecular and functional diversity of kinesin proteins
were treated with gabapentin (30 mg/kg i.p). For open field, rota-rod
[15,19,20]. Hence, in the present study, we have examined the effect of
and place preference studies we used another four group of nerve
tozasertib, a pan-Aurora kinase inhibitor, on the KIF17-NR2B crosstalk
injured rats which were treated with vehicle, tozasertib (40 mg/kg i.p),
in dorsal root ganglion and spinal cord of nerve injured rats and its effect
gabapentin (30 mg/kg i.p) and morphine (10 mg/kg i.p) respectively.
on neuropathic pain.

2. Methodology 2.5. Animal behavior tests

2.1. Drugs and reagents 2.5.1. Thermal stimuli pain assays

Tozasertib (VX-680) was purchased from Wuhan Goldenwing, China, 2.5.1.1. Tail flick test: Analgesic assay. The test was performed to check
and was reconstituted in 1% dimethyl sulfoxide (DMSO), 30% poly­ the effect of tozasertib on the thermal threshold in naïve rats [23]. Rats
ethylene glycol (PEG) 400, and 49% saline. Gabapentin and morphine were restrained (Ugo Basile) and a thermal source was applied to the
sulfate were procured from Sigma Aldrich (USA) and Sir SunderlLal tail. The response time was measured using a detector device (Ugo
Hospital Pharmacy, Banaras Hindu University, Varanasi, India. Both the Basile, Italy). The cut-off time was set at 10 sec. The heat intensity was
drugs were reconstituted in saline for i.p. administration. cDNA Syn­ standardized by maintaining the average response time between 3 and 4
thesis kit (#K1622) and Maxima SYBR Green/Fluorfescein qPCR Master s.
Mix (#K0241) were purchased from thermo scientific, USA. KIF17
antibody (sc-137040) was purchased from Santacruz whileanti- 2.5.1.2. Hargreaves Test: Thermal hyperalgesia. Thermal hyperalgesia
NMDAR2B (ab28373) and NFκβ antibodies (ab7971) were purchased was measured in nerve injured rats by using Hargreaves apparatus (Ugo
from Abcam, UK. Secondary antibodies, rabbit anti-mouse IgG H&L Basile, Italy). The procedure was performed in accordance with the
(HRP) (ab6728) and goat anti-rabbit IgG H&L (HRP) (ab6721) were also previously published study [24,25]. Animals were enclosed in trans­
purchased from Abcam, UK. parent acrylic chambers and allowed to acclimatized on Hargreaves
apparatus. On test day heat was applied using an automated infrared
2.2. Experimental animals (IR) beam source and cut off time was set at 20 s. The heat was applied to
the plantar surface of the hind paw and readings of both ipsilateral and
Male Sprague Dawley rats (200–250 gm) were used in the study. contralateral paws were taken in triplicate. All the experimental groups
Animals were housed four per cage in standard housing conditions; were tested for thermal hyperalgesia pre-injury baseline followed by
temperature 21 ± 2 ◦ C; 12-hour light–dark cycle. Animals were provided pre-drug baseline and at different time-points post drug administration.

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A. Uniyal et al. International Immunopharmacology 106 (2022) 108622

2.5.2. Static and dynamic mechanical allodynia test 2.5.5. Conditioned place preference: Spontaneous ongoing pain assay
A three-chambered (A, B, and C) conditioned place preference (CPP)
2.5.2.1. von-Frey hair test: Static allodynia. von-Frey hair test was used apparatus [22,30,31] where C was the central corridor section giving
to assess the dynamic mechanical allodynia in nerve injured rats. The entries to the adjacent chambers A and B, both with different visual and
mechanical sensitivity was assessed using von-Frey filaments of different tactile cues for the nerve injured rats treated with vehicle or drug. We
intensities ranging from 0.40 g (gm) to 13 gm [26]. Animals were placed used Any Maze software (Version 7.0 Stoelting, USA). to video-track and
in the plexi-glass chambers on the top of wire-mesh table and the von- analyse the time duration spent by each rat in different chambers. An­
Frey filaments were applied in the subplantar region of rats paw from imals were habituated in the test room for 30 mins followed by accli­
the bottom of the mesh. The up-down method was used to measure the matization in the CPP apparatus for 15 min. On the next day,
mechanical sensitivity before and after nerve injury and post-drug preconditioning baselines were video recorded for each rat to measure
treatment in rats. von-Frey hairs were applied at the middle of sub- their pre-drug compartment preference. During preconditioning, ani­
plantar region of hind paw of rats. Paw withdrawal, licking, and mals were allowed to freely explore all the compartments and video
shaking of the paw in response to von-Frey filament was considered as a tracking was done for 15 mins to find out the time spent by rats in each
positive response. The test was started with a lower force of 1.8 gm chamber. Rats showing preferences <20% and more than 80% for any of
filament. If there was no response, then filament of higher force was the chambers were excluded from the test. Further, conditioning was
used. When the response was positive then testing with lower force done for two days involving two sessions. In the morning session animals
filament was done. The test was performed till five positive responses were administered with vehicle and restricted in the paired chamber for
were observed or the test reaches the withdrawal maximum and the a period of 30 mins. Four hours later, second session was performed
minimum threshold at higher or lower forces. Pre-injury baseline and where animals were given drug and restricted to the paired chamber for
0.5 hr, 1 hr, 2 hr, 4hr and 24 hr post-drug/vehicle administration 30 mins. On trial day, post-conditioning was performed where rats were
thresholds were measured in nerve injured rats. gently placed in the corridor and were allowed to freely move across all
the compartments. The post-conditioning trial was video- recorded for
2.5.2.2. Cotton swab test: Dynamic mechanical test. Cotton swab test was 15 mins and time spent by rats in each chamber was measured.
performed to assess the dynamic mechanical allodynia in rats after nerve
injury and at different time points post-drug treatment [27]. Rats were 2.5.6. Behavioral neurotoxicity assays
kept in plexiglass chambers placed on elevated mesh apparatus. Using
cotton swab, the hind paws of rats were gently stroked. Latency of the 2.5.6.1. Rota-rod test. Rota-rod test was performed to measure the
paw withdrawal was recorded with a cut-off time of 15 sec. Care was motor-coordination activity of rats before and after drug treatment. The
taken while performing this test so as to avoid considering the normal test was performed in accordance with previous studies [26,32]. On the
movement of the rat. first day, rats were trained on rota-rod apparatus for 120 sec at the lower
speed of 5 revolution per minute (rpm). On the trial day, the perfor­
2.5.3. Cold stimuli pain assay mance of rats was measured before and after drug administration at a
constant speed of 25 rpm. Cut off time was set at 120 s and the time for
2.5.3.1. Cold plate test. Rats were placed in a clear plexiglass chamber which rats remained on the accelerating rod was noted down as time
with an ice floor [28]. A temperature sensor was placed on the surface to spent on rota-rod.
monitor the temperature changes. The temperature was maintained at
4 ◦ C ± 2 ◦ C. Moderate illumination was made for the whole duration of 2.5.6.2. Open field test. The test was performed to evaluate the effect of
the test. The test was video recorded for 1 min and scoring of paw la­ the drugs on the locomotor behavior of rats [31,33]. The method used
tency or licking was measured by a blind observer. Pre-injury baseline has been described in our previously published studies [31]. Habituation
and 0.5 hr, 1 hr, 2 hr, 4hr and 24 hr post-drug/vehicle administration was performed one day before the test in order to make the rats familiar
paw lifts were counted in nerve injured rats. with the novel environment. On testing day, rats were placed in the open
filed apparatus (45x45x75cm) and the session was video recorded for
2.5.3.2. Acetone evaporation test. The cold allodynia was measured by 10 min. Parameters such as total distance travelled and average speed
the acetone evaporation cooling test. Rats were acclimatized to the was measured using Any Maze software (Version 7.0 Stoelting, USA).
plexiglass chambers kept on Von-Frey mesh. A drop of acetone (100 μl)
was applied to the dorsal surface of the hind paw without touching the
skin. Three trials were performed to evaluate the cold allodynia. The test 2.6. Molecular studies
was video recorded for 1 min and scored by a blind observer. Scoring
was graded in four-point response 0, no response; 1, flicking of the paw; 2.6.1. Tissue harvesting and storage
2, repeated flicking of the paw; 3, repeated paw flicking with licking Euthanasia was performed after the completion of the behavioral
[29]. studies and the animals were sacrificed humanely using isoflurane
anaesthesia followed by cervical decapitation. Ipsilateral and contra­
2.5.4. Short nociceptive stimuli: lateral lumber L4-L5 dorsal root ganglion were dissected out by cutting
through the muscles and pulling out the transverse processes of the
spinal cord [15,22]. Further, the deep cut was made through the skin
2.5.4.1. Tail clip test. Alligator clips were applied to the tail of rats [16].
above the spinal cord. The vertebral column was removed using a ron­
The latency of the rat to reach the clip by mouth was considered as the
geur and dumont forceps and the spinal cord was exposed fully. The L4-
end point. Generally, animals reach and start licking the tail in 3–4 sec.
L5 lumbar region was identified and cut from the middle section to
Further, the effect of the drug was observed if it can increase the
divide the ipsilateral and contralateral spinal cord into two equal halves.
threshold of pain in rats.
The sciatic nerve was also harvested by dissecting through the bicep
femoris muscle. All tissues were stored at − 80 ◦ C for further biochemical
2.5.4.2. Pinprick test. A 22-gauge needle was glued on the von-Frey hair
and molecular analysis.
[16]. The pin was applied to the hind paw of rats. Care was taken while
applying the pin to the paw to avoid puncture of the skin. Ten trials were
2.6.2. Reverse transcription polymerase chain reaction (rtPCR) analysis
performed for each paw. The frequency of paw withdrawal per ten trials
Total RNA was isolated from the spinal cord and DRG tissues using
was recorded by a blind observer.
the Trizol reagent method. Further, the cDNA was prepared using a

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A. Uniyal et al. International Immunopharmacology 106 (2022) 108622

cDNA Synthesis kit (K1622 Thermo scientific). A reaction master mix of injury induced-thermal hyperalgesia. A significant effect on thermal
15 µl was prepared by mixing Maxima SYBR Green/Fluorescein qPCR hyperalgesia was observed across the groups [F (5, 42) = 322; p <
Master Mix (K0241), forward primer, reverse primer, template DNA, 0.001] and time points [F (5.03, 211) = 304; p < 0.001]. Moreover,
and nuclease-free water (Sigma W4502). The run was performed on contralateral PWL (Fig. S1A) of nerve injured rats were not altered
Rotor-Gene Q (Qiagen, Germany). Table S1 (supplementary file) shows before and after, CCI surgery and drug treatments (tozasertib and
the list of used primers for rtPCR. gabapentin).
The standard drug gabapentin (30 mg/kg i.p) showed significant
2.6.3. Western blotting effect at 30 min post-administration (p = 0.04), 1 hr (P < 0.001), 2 hr (p
Tissue lysate was prepared in radioimmunoprecipitation assay buffer < 0.001), and the effect lasted for the 4-hr post-administration (p <
(RIPA) buffer using an automated hand homogenizer [15,34]. Further, 0.001) as compared to the vehicle treated nerve injured rats.
incubation was performed on ice followed by centrifugation at 16,000g
at 4 ◦ C. Protein estimation was performed using Bradford reagent. An 3.1.2. Tozasertib treatment inhibits mechanical allodynia (static but not
equal amount of protein was loaded and made run on a sodium dodecyl dynamic) and hyperalgesia in nerve-injured rats
sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) setup. The Mechanical allodynia and mechanical hyperalgesia are one of the
transfer of protein from gel was done on a Poly(vinylidene fluoride) prominent symptoms in patients suffering from neuropathic pain. We
(PVDF) membrane. PVDF membrane was blocked with 8% milk for 2 hrs tested the effect of tozasertib on both static and dynamic mechanical
followed by incubation with primary antibodies of KIF17 (1:1000), allodynia by using von-Frey hair test and cotton bud test respectively.
NR2B (2 µg/ml), and beta-actin (0.5 µg/ml) at 4 ◦ C for overnight. We observed a significant effect across the groups [F (5, 42) = 133; p <
Membranes were washed with tris-buffered saline with 0.01% tween 20 0.001] and time points [F (4.00, 168) = 230; p < 0.001] in paw with­
(TBST) and incubated with respective secondary antibodies for 2 hrs at drawal threshold (PWT) of nerve injured and drug treated rats in von-
room temperature. Enhanced chemiluminescence substrate (Biorad, Frey hair test. We found a significant decrease in static allodynia as
USA) was used to detect the immune complexes. Blots were visualized ipsilateral paw withdrawal thresholds at day 14 post-CCI surgery were
using ChemiDoc™, BioRad, Hercules, California, United States. Finally, significantly decreased as compared to their pre-injury baselines and
the quantification of blots was done using image J software. naïve group rats (p < 0.001) (Fig. 1B). Tozasertib treatment (10, 20 and
40 mg/kg i.p) significantly restored the decreased paw withdrawal
2.6.4. Statistical analysis threshold of nerve injured rats in a dose-dependent manner. At 1 hr post-
Behavioral data was analyzed using one-way analysis of variance administration, tozasertib (10 mg/kg) (p = 0.002), 20 mg/kg (p =
(ANOVA) followed by Tukey’s multiple comparison test and two-way 0.010) and 40 mg/kg (p = 0.001) showed a significant effect as
ANOVA followed by Bonferroni’s multiple comparisons. For two-way compared to their pre-drug baselines and vehicle treated nerve injured
ANOVA we used a repeated measure model of statistical analysis. rats. The peak anti-allodynic effect was observed at 2 hr (10 mg/kg p =
Microsoft excel was used to interpret the raw data and calculate the 0.002; 20 and 40 mg/kg p < 0.001) post- tozasertib administration
mean, standard error mean (SEM), % maximum possible effect (MPE), which lasted for 4 hrs (10 and 20 mg/kg, p = 0.04; 40 mg/kg p = 0.003).
fold change for rtPCR and western blot data. Molecular studies data was The % MPE data suggested that tozasertib showed a dose-dependent
analyzed by using one-way ANOVA followed by Tukey’s multiple effect at 10 mg/kg, 20 mg/kg, and 40 mg/kg doses (Fig. 1B). Gaba­
comparison test. GraphPad Prism 8.0 was used to perform the statistical pentin (30 mg/kg i.p) treatment also attenuated the mechanical allo­
analysis. Data was presented as mean ± SEM. P < 0.05 was considered dynia in nerve injured rats as compared to the vehicle treated nerve
statistically significant. The sample size was calculated using Gpower injured rats. The contralateral PWT remains unaffected before and after,
statistical analysis tool and considering our previous studies. CCI induced nerve injury and treatment with tozasertib (10, 20 and 40
mg/kg i.p) and gabapentin (30 mg/kg i.p) (Fig. S1B).
3. Results Next, we examined the effect of tozasertib on the dynamic compo­
nent of the mechanical allodynia using cotton swab test. Nerve injured
3.1. Effect of tozasertib on nerve injury-induced pain-like behavior in rats rats developed significant [F (5, 42) = 118; P < 0.0001) dynamic me­
chanical allodynia after CCI surgery as evident by the decrease in paw
3.1.1. Tozasertib treatment attenuates thermal hyperalgesia in nerve withdrawal latency post non-noxious stimuli application as compared to
injured rats their pre-CCI baseline and naïve rats (P < 0.001). Interestingly, toza­
We have performed a battery of pain behavioral assays ranging from sertib (10, 20, and 40 mg/kg i.p) treatment did not showed inhibition of
thermal, mechanical, cold to spontaneous ongoing pain to assess the dynamic mechanical allodynia in nerve injured rats (Fig. 1C). However,
efficacy of pan aurora kinase inhibition on neuropathic pain. Chronic we did not observe a significant effect on dynamic mechanical allodynia
constriction injury (CCI) induced nerve injury led to significant decrease before and after, nerve injury and drug treatments (tozasertib and
in ipsilateral paw withdrawal latencies (PWL) of nerve injured rats (p < gabapentin) as compared to their pre-injury and pre-drug baselines.
0.001) as compared to their pre injury baselines and healthy naïve group The mechanical hyperalgesia was assessed using the pinprick test.
(Fig. 1A). Tozasertib treatment (10, 20, and 40 mg/kg i.p) significantly We have observed a significant effect across the groups (p < 0.01) in
attenuates the thermal hyperalgesia as evident by an increase in PWL of paw withdrawal frequency (PWF) of nerve injured and drug-treated rats.
nerve injured rats as compared to their pre-drug baselines. An increase Nerve injured rats showed a significant (p < 0.001) increase in ipsilat­
in latency trend was observed at 30 min post-tozasertib administration eral PWF as compared to their pre-injury baselines and naïve rats.
which was absent in vehicle treated nerve injured rats (Fig. 1A). The Tozasertib treatment significantly decreased the PWF of nerve injured
significant effect of tozasertib started at 1 hr post-administration i.e 10 rats at 1 hr (20 mg/kg, p = 0.01; and 40 mg/kg p = 0.002), 2 hr (p <
mg/kg (p = 0.002), 20 mg/kg (p < 0.001) and 40 mg/kg (p < 0.001). 0.001) and 4 hr (20 mg/kg, p = 0.02 and 40 mg/kg p = 0.03) post drug-
The peak therapeutic effect of tozasertib 10, 20, and 40 mg/kg (p < administration as compared to the vehicle treated group. However, 10
0.001) was observed at 2 hr post-drug administration which was lasted mg/kg tozasertib did not produced significant inhibition of mechanical
up to 4 h followed by a decline in the effect of tozasertib. However, 10 hyperalgesia specifically at 1 hr and 4 hr post-drug administration
mg/kg dose did not show significant effect at 4 hr post-drug adminis­ (Fig. 1D). Gabapentin (30 mg/kg i.p) treated nerve-injured rats showed a
tration in restoring nerve injury-induced decreased PWL. The maximum significant increase in PWF at different time points as compared to the
possible effect of tozasertib was found to be around 100% at 2 hrs with vehicle treated nerve injured rats. There was no significant effect
the dose of 40 mg/kg (Fig. 1A). The percentage MPE in Fig. 1A has observed in contralateral PWL before and after CCI induced nerve injury
suggested the dose-dependent effect of tozasertib in attenuation of nerve and treatment with tozasertib (10, 20 and 40 mg/kg i.p) and gabapentin

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A. Uniyal et al. International Immunopharmacology 106 (2022) 108622

Fig. 1. Effect of pan-Aurora kinase inhibition on pain-like behavior in nerve-injured rats. (A) Hargreaves test: Nerve injury significantly decreased the paw
withdrawal latency (PWL) of rats on day 14 post-CCI surgery as compared to their pre-injury baseline. Tozasertib (10, 20 and 40 mg/kg i.p) and gabapentin (30 mg/
kg i.p) administration resulted in a significant and dose-dependent increase in PWL of nerve injured rats as compared to their pre-drug baseline. (B) von Frey hair
test: Nerve injury-induced significant decrease in paw withdrawal threshold (PWT) of rats which was significantly restored in a dose-dependent manner on treatment
with tozasertib (10, 20 and 40 mg/kg i.p) and gabapentin (30 mg/kg i.p). (C) Cotton swab test: CCI-induced nerve injury significantly decreased paw withdrawal
latency (PWL) in rats as compared to their pre-CCI baseline. Gabapentin (30 mg/kg i.p) but not tozasertib (10, 20 and 40 mg/kg i.p) treatment led to significant
increase in PWL of nerve injured rats as compared to their pre-treatment baseline. (D) Pinprick test: CCI-induced nerve injury resulted in increased paw withdrawal
frequency (PWF) to noxious mechanical stimuli in rats which was significantly attenuated on treatment with tozasertib (10, 20 and 40 mg/kg i.p) and gabapentin (30
mg/kg i.p) as compared to their pre-drug baseline. (E) Acetone test: Nerve injury-induced cold hypersensitivity on day 14-post CCI surgery in rats which was
significantly attenuated by tozasertib (10, 20 and 40 mg/kg i.p) and gabapentin (30 mg/kg i.p). (F) Cold hyperalgesia test: CCI surgery-induced cold hyperalgesia in
rats in response to noxious cold stimuli. Treatment with tozasertib (10, 20 and 40 mg/kg i.p) and gabapentin (30 mg/kg i.p) significantly decreased number of paw
lifts in nerve injured rats as compared to their pre-drug baseline. Data were presented as mean ± SEM. ###P < 0.001 indicates statistical significance as compared to
the Naïve rats. *p < 0.05, **p < 0.01, and ***p < 0.001 indicates statistical significance as compared to the nerve injured rats. P < 0.05 was considered statistically
significant. Doses: Toza10, Tozasertib 10 mg/kg; Toza 20, Tozasertib 20 mg/kg; Toza40, Tozasertib 40 mg/kg; GP, Gabapentin 30 mg/kg. CCI, Chronic Constric­
tion Injury.

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(30 mg/kg i.p) (Fig. S1C). Gabapentin (30 mg/kg i.p) also decreased the cold allodynia evident by
a significant decrease in ipsilateral paw response score at 0.5 hr (p =
3.1.3. Tozasertib attenuates cold allodynia and cold hyperalgesia in nerve 0.007), 1 hr (p < 0.001), 2 hr (p < 0.001) and 4 hr (p < 0.001) post
injured rats administration as compared to the vehicle treated CCI injured rats.
Hypersensitivity induced by non-noxious and noxious thermal Further we tested the effect of tozasertib on noxious cold stimuli (0◦ C)
stimuli is notable trait among neuropathic pain patient. Here we induced cold hyperalgesia. A significant effect on number of ipsilateral
investigated the effect of tozasertib on cold allodynia and cold hyper­ paw lifts was observed across the groups [F (5.28, 222) = 63.2; p <
algesia in nerve injured rats. Acetone drop test was used to evaluate the 0.001] and time point [F (5, 42) = 74.3; p < 0.001] in vehicle and drug
non-noxious stimuli evoked cold allodynia in CCI injured rats. Two-way administered nerve injured rats. We have found that CCI induced nerve
ANOVA followed by Bonferroni’s multiple comparison suggested a sig­ injury has increased the number of ipsilateral paw lifts significantly in
nificant effect across the groups [F (5, 42) = 173; P < 0.001] and time presence of noxious cold stimulus as compared to their pre-injury
points [F (4.75, 199) = 155; P < 0.001] in response score of vehicle and baselines and naïve rats (p < 0.001) (Fig. 1F). There was a significant
drug treated nerve injured rats. CCI injury has significantly increased the reduction in number of paw lifts post tozasertib administration at 1 hr
response score to non-noxious cold stimuli in ipsilateral paw as (10 mg/kg, p = 0.02; 20 mg/kg, p = 0.01 and 40 mg/kg < 0.001), 2 hr
compared to the respective pre-injury baselines and naïve rats (p < (10 mg/kg, p = 0.04; 20 mg/kg, p = 0.001 and 40 mg/kg p < 0.001) and
0.001). Tozasertib treatment significantly decrease the response score at 4 hr (10 mg/kg, p = 0.02; 20 mg/kg, p = 0.008 and 40 mg/kg, p =
1 hr (10 mg/kg, p = 0.002; 20 and 40 mg/kg, p < 0.001), 2 hr (p < 0.001) as compared to the vehicle treated nerve injured rats. Gabapentin
0.001) and 4 hr (10 mg.kg, p = 0.02; 20 mg/kg, p = 0.009 and 40 mg/ also significantly decreased the number of ipsilateral paw lifts as
kg, p < 0.001) post administration as compared to their respective pre- compared to the CCI injured rats post 0.5 hr (p = 0.002), 1 hr (p <
treatment baselines and vehicle treated nerve injured rats (Fig. 1E). 0.001), 2 hr (p < 0.001) and 4 hr (p < 0.05) of administration. However,

Fig. 2. Effect of tozasertib on normal pain threshold of healthy naïve rats. (A and B) Tail flick test: Tozasertib (10, 20 and 40 mg/kg i.p) and gabapentin (30
mg/kg i.p). treatment showed no significant effect on normal pain threshold of naïve rats in response to noxious thermal stimuli. However, morphine (10 mg/kg i.p)
treatment significantly increased the baseline tail-flick latency of naïve rats. (C and D) Tail clip test: Tozasertib (10, 20 and 40 mg/kg i.p) and gabapentin (30 mg/kg
i.p).treatment did not alter the baseline threshold value of naïve rats in response to noxious mechanical stimuli. However, morphine (10 mg/kg i.p) treatment
significantly increased the latency to attack in healthy naïve rats. Data were presented as mean ± SEM. ###P < 0.001 indicates statistical significance as compared to
the Naïve rats. *p < 0.05, **p < 0.01, and ***P < 0.001 indicates statistical significance as compared to the CCI rats. P < 0.05 was considered statistically significant.
Doses: Toza10, Tozasertib 10 mg/kg; Toza 20, Tozasertib 20 mg/kg; Toza40, Tozasertib 40 mg/kg; GP, Gabapentin 30 mg/kg. Naïve, Healthy rats.

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CCI induced nerve injury and drug treatments did not show a significant positive control. Both the test represents the hyperalgesic response to
effect on contralateral, allodynic response score and noxious stimuli noxious thermal and mechanical stimulus respectively. A two-way
induced cold hypersensitivity in rats as compared to their respective pre- ANOVA followed by Bonferroni’s multiple comparisons suggested that
injury and pre-drug baselines (Fig. S1 E and F). in the tail-flick test there was a significant effect across the groups [F (5,
42) = 33.5; p < 0.001] and time points [F (3.45, 145) = 3.64; p < 0.001]
in vehicle and drug treated nerve injured rats. We observed that the
3.2. Tozasertib did not altered the pain threshold of naïve uninjured rats morphine significantly increased tail-flick latency (Fig. 2A and B) as
compared to naïve rats (p < 0.001). The maximum possible effect post
To evaluate the effect of tozasertib on pain threshold in naïve rats we morphine administration was observed at 30 min (p < 0.001) and the
have performed the tail-flick and tail clip test with morphine as a

Fig. 3. Effect of tozasertib, a pan-Aurora kinase inhibitor, on spontaneous ongoing pain in nerve injured rats. (A, B and C) Conditioned place preference
(CPP) for tozasertib: Nerve-injured rats showed significant place preference behaviour in tozasertib (40 mg/kg i.p.) paired chamber as compared to the pre-
conditioning baseline. Interestingly, tozasertib (40 mg/kg i.p.) treatment did not produced CPP in healthy naïve rats. (D, E, and F) CPP for morphine:
Morphine (10 mg/kg i.p.) treatment significantly attenuates CCI-induced ongoing pain but also developed enhanced CPP in healthy naïve rats which indicates the
analgesic and addictive potential of morphine. (G, H and I) CPP for gabapentin: Gabapentin (30 mg/kg i.p.) treatment induced CPP in nerve injured rats but did not
produce place preference in healthy naïve rats. Data were presented as mean ± SEM. ###P < 0.001 indicates statistical significance as compared to the Naïve rats. *p
< 0.05, **p < 0.01, and ***p < 0.001 indicates statistical significance as compared to the CCI rats. P < 0.05 was considered statistically significant. Doses: Toza40,
Tozasertib 40 mg/kg; GP, Gabapentin 30 mg/kg. CCI, Chronic Constriction Injury.

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A. Uniyal et al. International Immunopharmacology 106 (2022) 108622

activity sustained up to 4 h (Fig. 2B). In the tail clip test, we observed a inhibitor tozasertib (10, 20, and 40 mg/kg i.p) did not altered the
significant effect post two-way ANOVA followed by Bonferroni’s mul­ nociceptive latency in both tail-flick and tail clip tests. Gabapentin
tiple comparisons across the groups [F (5, 42) = 50.1; p < 0.001] and treatment also did not alter the pain threshold in both the tests as
time points F (2.60, 109) = 18.9; p < 0.001] in vehicle and drug treated compared to the naïve rats. These findings suggest that tozasertib does
rats with CCI injury. Morphine increased the latency to noxious me­ not interfere with the normal pain thresholds of healthy naïve rats.
chanical stimulus significantly as compared to the naïve rats (p < 0.001)
(Fig. 2C). The significant effect of morphine persisted across the whole
duration of the test i.e., 4 hrs (Fig. 2D). However, the pan-Aurora kinase

Fig. 4. Effect of tozasertib on locomotor activity of nerve injured rats. (A, B and C) Open field test: Tozasertib (40 mg/kg i.p.) and gabapentin (30 mg/kg i.p.)
treatment did not affect the locomotor activity of nerve injured rats in open field arena as compared to the vehicle treated rats. However, morphine (10 mg/kg i.p.)
treated rats showed a significant decline in total distance travelled and average speed. (A) Open field track plots of nerve injured rats treated with vehicle, tozasertib,
gabapentin and morphine. (B) Total distance travelled by nerve injured rats after treatment with vehicle, tozasertib, gabapentin and morphine. (C) Average speed of
nerve injured rats in open field arena after treatment with vehicle, tozasertib, gabapentin and morphine. (D) Rotarod test: Tozasertib (40 mg/kg i.p.) and gabapentin
(30 mg/kg i.p) treated rats did not show significant decrease in fall-time in rota-rod test as compared to their pre-treatment baseline. However, morphine (10 mg/kg i.
p.) treatment significantly decreased fall-time of rats as compared to the pre-morphine baseline. Data were presented as mean ± SEM. ###p < 0.001 indicates
statistical significance as compared to the Naïve rats. *p < 0.05, **p < 0.01, and ***P < 0.001 indicates statistical significance as compared to the CCI rats. P < 0.05
was considered statistically significant. Doses: Toza40, Tozasertib 40 mg/kg; GP, Gabapentin 30 mg/kg. CCI, Chronic Constriction Injury.

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3.3. Tozasertib inhibits spontaneous ongoing pain behavior in nerve- compared to the naïve rats (Figs. 5 and 6). Tozasertib treatment signif­
injured rats icantly decreased the ipsilateral L4-L5, spinal and DRG expression of
NR2B mRNA and protein at 20 mg/kg (p < 0.001) and 40 mg/kg (p <
In neuropathic pain patients, apart from the sensory hyper-sensation 0.001) doses as compared to the vehicle treated nerve injured rats.
the emotional pain components also deteriorate the quality of life. Gabapentin treatment also decreased the NR2B mRNA and protein
Ongoing pain is a challenging clinical problem that persist in patients expression in both DRG tissues (p < 0.001) and spinal cord (p < 0.001)
with chronic pain condition. We have performed the Conditioned place as compared to the vehicle treated nerve injured rats (Figs. 5 and 6). It
preference (CPP) paradigm to assess the effect of tozasertib on sponta­ indicates that both tozasertib and gabapentin may involve the NR2B
neous ongoing pain-like behavior in rats (Figs. 3 and S2). Tozasertib (40 mediated mechanism during the attenuation of nerve injury-induced
mg/kg i.p) significantly (p < 0.001) attenuates ongoing pain in nerve- neuropathic pain. We did not observe a significant on contralateral
injured rats as evident from increase in preference to the tozasertib- L4-L5, DRG and spinal NR2B expression in nerve injured rats and drug
paired chamber as compared to the vehicle-paired chamber during treated rats (tozasertib and gabapentin) as compared to the naïve rats
post-conditioning trial (Fig. 3 A and B). Moreover, there was a signifi­ (Fig. S3).
cant (p < 0.001) increase in difference score in tozasertib paired
chamber as compared to the vehicle paired chamber in nerve injured 3.5.2. Tozasertib but not gabapentin suppressed the nerve injury-induced
rats (Fig. 3C). Interestingly, tozasertib (40 mg/kg i.p) did not induce CPP KIF17 expression
in naïve rats as there was no significant change in preference to the KIF17 nanomotor is the major trafficking partner of NR2B subunit to
tozasertib v/s vehicle paired chamber during post-conditioning trial make the NMDAR functionalized into the synaptic membrane. We
(Fig. 3B). This suggest that tozasertib induced CPP is dependent on relief observed that CCI injury increased the expression of KIF17 mRNA and
of pain-induced due to nerve injury and the molecule does not suffer protein in ipsilateral L4-L5, DRG [F (5, 18) = 20.1; p < 0.001 and F (5,
from abuse liability potential. Furthermore, we have found that gaba­ 12) = 14.5; p < 0.001 respectively] and spinal cord [F (5, 18) = 18.1; p
pentin treatment also induced significant (p < 0.01) place preference < 0.001 and F (5, 12) = 9.77; p < 0.001 respectively] tissues of rats as
behavior exclusively in nerve injured rats only (Fig. 3G and H). How­ compared to the naïve rats. Tozasertib treatment suppressed the ipsi­
ever, morphine treatment induced a significant place preference lateral L4-L5, DRG and spinal expression of KIF17 mRNA and protein at
behaviour in both nerve injured and naïve rats (p < 0001) pointing 20 mg/kg (p < 0.01 and respectively p < 0.001) and 40 mg/kg (p <
towards its analgesic and addictive properties. 0.001) as compared to the vehicle treated nerve injured rats (Figs. 5 and
6). Gabapentin (30 mg/kg i.p) treatment did not produce any significant
3.4. Tozasertib does not affect locomotor or exploratory activity of nerve effect on the KIF17 expression in ipsilateral L4-L5, DRG and spinal cord
injured rats tissues as compared to the vehicle treated nerve injured rats. From these
findings, we conclude that inhibition of aurora kinase enzyme interferes
Further we investigated the effects of tozasertib on motor incoordi­ with KIF17-NR2B cargo system during neuropathic pain. Whereas,
nation and locomotor activity of rats. In the open field test, we observed gabapentin mediated pain relief does not involve the KIF17 mediated
that tozasertib (40 mg/kg i.p) treatment did not alter the exploratory or signaling in nerve injured rats. CCI induced nerve injury and, treatment
locomotor behavior of nerve injured rats (Fig. 4A, B, and C) as there was with tozasertib (10, 20 and 40 mg/kg i.p) and gabapentin (30 mg/kg i.p)
no significant change in the total distance travelled and average speed of did not show a significant effect on contralateral L4-L5, DRG and spinal
tozasertib treated nerve injured rats as compared to the naïve rats. expressions of KIF17 protein as compared to the naïve rats (Fig. S3).
Moreover, treatment with gabapentin (30 mg/kg i.p) also produced no
observable effects on the locomotor behavior of nerve injured rats. 3.6. Tozasertib treatment attenuates inflammatory signaling in nerve
However, morphine (10 mg/kg i.p) administration led to significant (p injured rats
< 0.05) impairment in the locomotor activity of rats as compared to its
pre-treatment baseline and vehicle treated nerve injured rats. In rotarod Inflammatory cytokines are the key modulatory mechanisms behind
test also, tozasertib (40 mg/kg i.p) as well as gabapentin (30 mg/kg i.p) the development of pain pathophysiology. In chronic pain condition
treatment showed no significant change in time spent on rod as activation of NMDA stimulates release of cytokines that further pro­
compared to the nerve injured rats (Fig. 4D). While morphine treatment motes the pathophysiology of the disorder. One-way ANOVA followed
significantly reduced (p < 0.001) the fall latency of rats in rota-rod test by Tukey’s multiple comparison test suggested a significant effect across
as compared to its pre-treatment baseline pointing towards its sedative the groups on mRNA and protein levels of ipsilateral L4-L5, DRG [F (5,
properties. 12) = 6.98; p < 0.01 and F (5, 18) = 10.2; P < 0.001 respectively] and
spinal [F (5, 18) = 28.6; P < 0.001 and F (5, 12) = 6.98; p < 0.01
3.5. Tozasertib interferes with KIF17 and NR2B expression in the spinal respectively] tissue of vehicle and drug treated nerve injured rats. CCI-
cord and dorsal root ganglion of nerve-injured rats induced nerve injury significantly increased the nuclear factor kappa β
(NFκβ) mRNA and protein expressions in L4-L5, DRG (p < 0.001 and p <
3.5.1. Tozasertib significantly reduced nerve injury induced-NR2B mRNA 0.01 respectively) and spinal cord (p < 0.001 and p < 0.01 respectively)
and protein expression in spinal and DRG tissue tissues which was significantly attenuated on treatment with tozasertib
NR2B plays an important role in the development of central sensi­ at 10 mg/kg (p < 0.05 and p < 0.01 respectively), 20 mg/kg (p < 0.001
tization during chronic pain condition. rtPCR and western blot studies and p < 0.01 respectively), and 40 mg/kg (p < 0.001) (Fig. 7). Further,
were conducted to investigate the effect of aurora kinase inhibition on we measured the mRNA levels of inflammatory cytokines such as
mRNA and protein expression of NR2B in spinal cord and DRG tissues of interleukin-6 (IL-6), interleukin 1 β (IL1β), and tumour necrosis factor-α
nerve injured rats. Using one-way ANOVA followed by Tukey’s multiple (TNF-α) in ipsilateral L4-L5, DRG and spinal cord tissues of rats. One-
comparison test, we found a significant effect across the groups on both way ANOVA followed by Tukey’s multiple comparison demonstrated a
mRNA and protein expression of NR2B in ipsilateral L4-L5, DRG [F (5, significant effect across the groups on DRG tissues and spinal cord IL-6
18) = 34.7; p < 0.001 and F (5, 12) = 15.3; p < 0.001 respectively] and [F (5, 18) = 9.66; p < 0.001 and F (5, 18) = 9.64; p < 0.001 respec­
spinal cord [F (5, 18) = 30.7; p < 0.05 and F (5, 12) = 14.1; p < 0.001 tively], IL1β [F (5, 18) = 11.1: p < 0.001 and F (5, 18) = 16.4; p < 0.001
respectively] tissues of vehicle and drug treated nerve injured rats. We respectively] and TNF-α [F (5, 18) = 20.9; p < 0.001 and F (5, 18) =
found that nerve injury has significantly increased the mRNA and pro­ 15.0; p < 0.001 respectively] levels in vehicle and drug treated nerve
tein expression of NR2B in the DRG (p < 0.001 and p < 0.01 respec­ injured rats (Fig. 8). Nerve injury-induced significant increase in ipsi­
tively) and spinal cord (p < 0.001 and p < 0.01 respectively) as lateral L4-L5, DRG and spinal expression of IL-6 (p < 0.001), IL1β (p <

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Fig. 5. Tozasertib decreased KIF17 and NR2B mRNA expressions in dorsal root ganglion (DRG) and spinal cord of nerve injured rats. (A) DRG mRNA
expression: CCI surgery significantly increased mRNA expressions of KIF17 (A1) and NR2B (A2) in ipsilateral L4-L5 DRG of rats which was significantly attenuated
by tozasertib treatment (20 and 40 mg/kg i.p.). Gabapentin (30 mg/kg i.p) treatment significantly reduced the ipsilateral L4-L5 DRG mRNA expression of NR2B but
does not have any effect on KIF17 mRNA expression. (B) Spinal mRNA expression: Nerve injury-induced significant increase in ipsilateral L4-L5 spinal expressions
of KIF17 and NR2B which was significantly decreased on treatment with tozasertib (20 and 40 mg/kg i.p.). While, gabapentin (30 mg/kg i.p) treatment significantly
decreased NR2B expression without affecting KIF17 expression in ipsilateral L4-L5 spinal cord of nerve injured rats. Data were presented as mean ± SEM. ###p <
0.001 indicates statistical significance as compared to the Naïve rats. *p < 0.05, **p < 0.01, and ***p < 0.001 indicates statistical significance as compared to the CCI
rats. p < 0.05 was considered statistically significant. n = 4 biological and n = 3 technical replicates. Doses: Toza10, Tozasertib 10 mg/kg; Toza 20, Tozasertib 20
mg/kg; Toza40, Tozasertib 40 mg/kg; GP, Gabapentin 30 mg/kg. CCI, Chronic Constriction Injury.

0.001), and TNF-α (p < 0.001) as compared to the naïve rats which was same is not effective in more than 70% of the patients on analgesic drug
significantly attenuated on treatment with different doses of tozasertib therapies [36]. Moreover, the drugs such as opioids in spite of being the
(10 mg/kg, 20 mg/kg and 40 mg/kg). Gabapentin (30 mg/kg) treatment gold-standard therapy for the treatment of neuropathic pain has been
also reduced the mRNA expression of IL-6 (p < 0.001) IL1β (p < 0.001), degraded to fourth line of therapy from first and second line because of
and TNF-α (p < 0.001) in DRG and spinal cord tissues of nerve injured their severe side effects including but not limited to sedation, motor
rats as compared to the vehicle treatment. incoordination, constipation, respiratory depression and their abuse li­
ability [37]. Therefore, there is any urgent need to develop the novel
4. Discussion pain-killers which do not produce the classical side effects associated
with opioids and other non-opioid analgesics. Recent studies has sug­
The somatosensory nervous system is involved in the processing of gested the involvement of KIF17 nanomotor in pathophysiology of
different stimuli (touch, temperature, vibration, etc.) [35] and plays an chronic pain [16–18]. Targeting KIF17 mediated trafficking could pro­
important role in the transmission of different information including vide a novel insight towards the potential near future therapeutics for
pain signals. Neuropathic pain may occur due to a lesion or a disease in the treatment of neuropathic pain. Aurora kinases are among the key
the somatosensory nervous system and the treatment available for the enzymes that regulate the cell proliferation and cell division [38]. These

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Fig. 6. Inhibition of pan-Aurora kinase reduces elevated protein expressions of KIF17 and NR2B in dorsal root ganglion and spinal cord of nerve injured
rats. (A) DRG protein expressions (A1 and A2) Tozasertib treatment (20 and 40 mg/kg i.p.) attenuates CCI surgery-induced increase in protein expressions of KIF17
and NR2B in ipsilateral L4-L5 DRG of nerve injured rats. Whereas gabapentin (30 mg/kg i.p) treatment reduced only NR2B without affecting KIF17 protein ex­
pressions in ipsilateral L4-L5 DRG of nerve injured rats (B) Spinal protein expressions (B1 and B2) Nerve injury-induced significant increase in protein expressions
of KIF17 and NR2B in ipsilateral L4-L5 spinal cord tissues of rats which was significantly attenuated on treatment with tozasertib (20 and 40 mg/kg i.p.). While,
treatment with gabapentin (30 mg/kg i.p) significantly reduced the spinal expressions NR2B without affecting KIF17 levels in nerve injured rats. Data were presented
as mean ± SEM. ###p < 0.001 indicates statistical significance as compared to the Naïve rats. *p < 0.05, **p < 0.01, and ***P < 0.001 indicates statistical sig­
nificance as compared to the CCI rats. p < 0.05 was considered statistically significant. n = 3. Doses: Toza10, Tozasertib 10 mg/kg; Toza 20, Tozasertib 20 mg/kg;
Toza40, Tozasertib 40 mg/kg; GP, Gabapentin 30 mg/kg. CCI, Chronic Constriction Injury.

kinases also phosphorylate the mitotic centrosome-associated kinesins syndromes and involves both static and dynamic component [35]. The
and regulate the synthesis of a variety of molecular switches associated Aδ and C fibers regulates the static mechanical allodynia, whereas, the
with cell signaling [38]. During chronic pain conditions levels of aurora Aβ fibers are involved in the mediation of dynamic mechanical allodynia
kinase are elevated and are crucial for maintaining inflammatory [27,40]. We found that static mechanical allodynia was significantly
signaling at spinal and supra-spinal levels [39]. Aurora kinase are blocked on treatment with Tozasertib without producing any inhibition
known to be involved in the synthesis and activation of kinesin proteins of dynamic allodynia in nerve-injured rats. Compounds such as
which may further regulate the downstream signaling involved in pain morphine that do not inhibit dynamic mechanical allodynia also known
processing after nerve injury. In the present study, we investigated the to exert their activity through Aδ and C fibers [41]. We also found that
effect of tozasertib, a pan-Aurora kinase inhibitor on nerve injury- tozasertib treatment does not interfere with the normal nociception in
induced neuropathic pain and the involvement of KIF17-NMDA cross­ healthy naïve rats unlike morphine and suggest the potential application
talk in the same. We found that pan-Aurora kinase inhibition leads to of this molecule during chronic pain condition. CNS side effects are the
significant and dose-dependent attenuation of thermal hyperalgesia most troublesome feature of most of the analgesics available in the clinic
(cold and heat) along with inhibition of static mechanical allodynia and for the treatment of neuropathic pain [26]. Therefore, we have decided
mechanical hyperalgesia in nerve-injured rats. Mechanical allodynia is to perform the in-vivo neurotoxicity assays using open field and rota-rod
one of the prominent feature of both neuropathic and postoperative pain test. Morphine treated rats showed significant decrease in total distance

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Fig. 7. Effect of tozasertib on NFkβ mRNA and protein expressions in dorsal root ganglion and spinal cord of nerve injured rats. mRNA expressions: CCI
induced-nerve injury increased mRNA expressions of NFkβ in in ipsilateral L4-L5, DRG (A) and spinal cord (B) of rats, which was significantly reversed on treatment
with tozasertib (10, 20 and 40 mg/kg i.p.) and gabapentin (30 mg/kg i.p). Protein expressions: Tozasertib (10, 20 and 40 mg/kg i.p.) and gabapentin (30 mg/kg i.p)
treatment significantly reduced nerve injury-induced induced protein expressions of NFkβ in ipsilateral L4-L5, DRG (C) and spinal cord (D) of nerve injured rats. Data
were presented as mean ± SEM. ###p < 0.001 indicates statistical significance as compared to the Naïve rats. *p < 0.05, **p < 0.01, and ***p < 0.001 indicates
statistical significance as compared to the CCI rats. p < 0.05 was considered statistically significant. For western blot n = 3 was used whereas for rtPCR n = 4
biological and n = 3 technical replicates were used. Doses: Toza10, Tozasertib 10 mg/kg; Toza 20, Tozasertib 20 mg/kg; Toza40, Tozasertib 40 mg/kg; GP,
Gabapentin 30 mg/kg. CCI, Chronic Constriction Injury.

travelled and average speed in open field test and decreased fall latency causing drug addiction.
as observe in rota-rod test. However, we did not observe any effect of NR2B subunit is involved in the development and maintenance of
tozasertib treatment at the highest tested dose (40 mg/kg i.p) on the chronic pain [42,43]. The surface expression of NR2B is higher in
locomotor and spontaneous exploratory activity of rats in the open field chronic pain which causes activation of several downstream pathways
test. The latency to fall in the rota-rod test was also not affected on including Ca++ signaling, protein kinase A (PKA), protein kinase C
treatment with Tozasertib (40 mg/kg i.p). (PKC), extracellular signal-regulated kinase (ERK), and mitogen acti­
Spontaneous ongoing pain is one of the important component of vated protein kinase (MAPK) pathways [4]. These multifarious path­
neuropathic pain and is increasingly being accepted as an parameter of ways cause central sensitization and enhanced pain transduction and
overall well-being in pain patients [35]. Treatment with tozasertib and perception. We have found that spinal and DRG expressions of NR2B
gabapentin produced a significant increase in place preference behav­ were increased after CCI-induced nerve injury in rats. Tozasertib treat­
iour indicating the amelioration of spontaneous ongoing pain in nerve ment significantly decreased the NR2B protein expression and mRNA
injured rats. However, both the drugs failed to induce-CPP in healthy levels in both spinal cord and DRG tissues of rats. Gabapentin treatment
naive rats suggesting that their place preference behaviour in CCI rats is also decreased the NR2B expression in the spinal and DRG tissues of
nerve injury dependent and points towards their non-addictive analgesic nerve-injured rats and the findings are in line with previous studies
potential. Whereas, morphine treatment leads to development of sig­ [44,45]. The anterograde trafficking of NR2B loaded cargo is mediated
nificant place preference behaviour in rats irrespective of their nerve through the KIF-17 nanomotor across the microtubule track. We have
injury state confirming its addictive potential. The present findings found that KIF-17 expressions were significantly increased in spinal cord
suggest that inhibition of aurora kinase produced inhibition of both and DRG of rats after CCI-induced nerve injury. This suggest that NR2B
evoked and spontaneous ongoing pain in nerve injured rats without subunit-dependent central sensitization might be mediated through KIF-

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Fig. 8. Effect of pan-Aurora kinase inhibition on nerve injury-induced inflammatory signaling in dorsal root ganglion and spinal cord of rats. (A) DRG
mRNA expressions: Nerve injury-induced significant increase in IL-6 (A1), IL1β (A2), TNF-α (A3) in ipsilateral DRG of rats which was significantly attenuated by
tozasertib (10, 20 and 40 mg/kg i.p) and gabapentin (30 mg/kg i.p) treatment. (B) Spinal mRNA expressions: Tozasertib (10, 20 and 40 mg/kg i.p) and gabapentin
(30 mg/kg i.p) treatment significantly attenuates CCI-induced increase in mRNA expressions of IL-6 (B1), IL1β (B2) and TNF-α (B3) in ipsilateral L4-L5 spinal cord of
rats. n = 4 biological and n = 3 technical replicates. Doses: Toza10, Tozasertib 10 mg/kg; Toza 20, Tozasertib 20 mg/kg; Toza40, Tozasertib 40 mg/kg; GP,
Gabapentin 30 mg/kg. CCI, Chronic Constriction Injury.

17 induced trafficking. Further, we have observed that tozasertib and central sensitization [46]. Inflammation occurs in response to any
treatment significantly decreased KIF17 mRNA expression and protein tissue injury or activation of immune system, and can itself lead to the
levels in both spinal cord and DRG tissues of nerve injured rats. Inhi­ lowering of nociceptive threshold. Proinflammatory cytokines such as
bition of KIF17 by tozasertib might be responsible for the attenuation of IL1β, IL-6 and TNF-α can modulate the nociceptive response by directly
NMDA mediated inflammatory signaling and decreased pain behaviour stimulating the nociceptors or by indirect pathway via prostaglandin
in nerve injured rats dependent signaling [47]. NMDARs by interacting with other tran­
The involvement of inflammatory mediators is a well-evident in the scriptional proteins can result in the release of various cytokines and
pathophysiology of neuropathic pain especially causing the peripheral promotes the development of neuropathic pain [48]. Moreover, the

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A. Uniyal et al. International Immunopharmacology 106 (2022) 108622

NMDA receptor system is linked with the activation of the NF-kβ Declaration of Competing Interest
pathway which promotes the transcription of several genes and activates
parallel cytokine signaling including, IL1β, IL-6, and TNF-α [49,50]. The authors declare that they have no known competing financial
Increased activity of these cytokines results in enhanced pain trans­ interests or personal relationships that could have appeared to influence
mission and central sensitization hereby, promoting the pathophysi­ the work reported in this paper.
ology of chronic pain [51]. Inhibition of aurora kinase is known to
regulate the inflammation via suppression of the mast cell activation, Acknowledgment
changes in macrophage morphology and spinal microgliosis [52]. In the
present work we found that tozasertib, a pan-Aurora kinase inhibitor We would also like to acknowledge the Indian Institute of Technol­
attenuates mRNA levels and protein expressions of NFκβ in spinal cord ogy, Banaras Hindu University, Varanasi, Uttar Pradesh, India for
and DRG tissue of nerve injured rats. Similar effects were observed with providing the necessary facilities and infrastructure.
mRNA levels of IL1β, IL-6, and TNF-α as these were found to be signif­
icantly suppressed by pan-Aurora kinase inhibition in both the DRG and Appendix A. Supplementary material
spinal cord. These results indicate toward the promising effect of toza­
sertib on neuroinflammation at spinal cord and DRG level of nerve Supplementary data to this article can be found online at https://doi.
injured rats. Several reports are suggesting the potential of anti- org/10.1016/j.intimp.2022.108622.
inflammatory compounds in variety of pain disorders, hence this ef­
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