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Duloxetine alloxan diabetic neuropathy

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Life Sciences 205 (2018) 113–124
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Life Sciences
journal homepage: www.elsevier.com/locate/lifescie
Neuroprotective effect of duloxetine in a mouse model of diabetic
neuropathy: Role of glia suppressing mechanisms
Mona K. Tawfika, Seham A. Helmyb,c, Dahlia I. Badrand, Sawsan A. Zaitonee,f,
T
⁎,1
a
Department of Pharmacology, Faculty of Medicine, Suez Canal University, Ismailia 41522, Egypt
Department of Cytology and Histology, Faculty of Veterinary Medicine, Suez Canal University, Ismailia 41522, Egypt
c
Female Collage of Applied Medical Sciences, Bisha University, Bisha, Saudi Arabia
d
Department of Biochemistry, Faculty of Medicine, Suez Canal University, Ismailia 41522, Egypt
e
Department of Pharmacology and Toxicology, Faculty of Pharmacy, University of Tabuk, Tabuk, Saudi Arabia
f
Department of Pharmacology and Toxicology, Faculty of Pharmacy, Suez Canal University, Ismailia 41522, Egypt
b
A R T I C LE I N FO
A B S T R A C T
Keywords:
Diabetic neuropathy
Duloxetine
Mouse
Sciatic nerve growth factor
Spinal astrocytes
Spinal microglia
Aims: Painful diabetic neuropathy (PDN) is one of the most frequent complications of diabetes and the current
therapies have limited efficacy. This study aimed to study the neuroprotective effect of duloxetine, a serotonin
noradrenaline reuptake inhibitor (SNRI), in a mouse model of diabetic neuropathy.
Main methods: Nine weeks after developing of PDN, mice were treated with either saline or duloxetine (15 or
30 mg/kg) for four weeks. The effect of duloxetine was assessed in terms of pain responses, histopathology of
sciatic nerve and spinal cord, sciatic nerve growth factor (NGF) gene expression and on the spinal expression of
astrocytes (glial fibrillary acidic protein, GFAP) and microglia (CD11b).
Key findings: The present results highlighted that duloxetine (30 mg/kg) increased the withdrawal threshold in
von-Frey test. In addition, both doses of duloxetine prolonged the licking time and latency to jump in the hotplate test. Moreover, duloxetine administration downregulated the spinal expression of both CD11b and GFAP
associated with enhancement in sciatic mRNA expression of NGF.
Significance: The current results highlighted that duloxetine provided peripheral and central neuroprotective
effects in neuropathic pain is, at least in part, related to its downregulation in spinal astrocytes and microglia.
Further, this neuroprotective effect was accompanied by upregulation of sciatic expression of NGF.
1. Introduction
The disease burden of diabetes mellitus (DM) is high and elevated
progressively worldwide [1]. Painful diabetic neuropathy (PDN) is the
most common chronic complication of DM, presented with variable
clinical manifestations [2,3]. It occurs as shooting pain in the form of
dysesthesias in the periphery of the limbs [4] and usually accompanied
by allodynia and hyperalgesia.
The pathogenesis of neuropathic pain is related to an interaction
between the immune system and the nervous system including the
spinal cord, the dorsal root ganglia, the peripheral nerves and the brain
as a result of glial cells activation in the CNS [5]. Astrocytes are the
most abundant glial cells representing 40%–50% of CNS glial cells [6].
Moreover, microglia had been recently involved in the pathogenesis of
neuropathic pain since they become activated upon injury [7]. Several
studies implicated upregulation of spinal cord microglia and astrocytes
⁎
1
in animal models of PDN [8–10]. Pain hypersensitization is sustained
by the activated astrocytes which overexpress the astrocytic marker: the
glial fibrillary acidic protein (GFAP), nitric oxide and other several
cytokines as interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α)
[11]. Management of PDN represents a great challenge for the clinicians due to either the failure of proper glycemic control in reducing
pain [12,13] or the use of ineffective drugs in controlling pain [14].
Multiple classes of drugs are extensively used trying to alleviate pain
resulting from PDN and improving the patient's quality of life, like nonsteroidal anti-inflammatory drugs, tricyclic-antidepressants with serotonin and noradrenaline reuptake blockage [15], antiepileptics including pregabalin or opioids (nonspecific analgesics) [16]. In addition,
trials to alleviate neuropathic pain in nerve-injured animal models by
blockage of microglial activation had been investigated in previous
studies using several drugs as minocycline [17–20] or the antidepressant fluoxetine [21].
Corresponding author at: Department of Pharmacology and Toxicology, Faculty of Pharmacy, Suez Canal University, Ismailia 41522, Egypt.
E-mail addresses: [email protected], [email protected] (S.A. Zaitone).
Current: Department of Pharmacology and Toxicology, Faculty of Pharmacy, University of Tabuk, Tabuk, Saudi Arabia.
https://doi.org/10.1016/j.lfs.2018.05.025
Received 7 December 2017; Received in revised form 26 April 2018; Accepted 11 May 2018
Available online 12 May 2018
0024-3205/ © 2018 Elsevier Inc. All rights reserved.
Life Sciences 205 (2018) 113–124
M.K. Tawfik et al.
determined for each mouse using a blood sample taken from the tail
vein using One Touch Ultra Mini glucometer (USA). Mice with fasting
blood glucose level > 250 mg/dl were considered diabetic and included
in the experiment.
Duloxetine ((+)-(S)-N-methyl-c-(1-naphthyloxy)-2-thiophenepropylamine) classified as a serotonin noradrenaline neuronal reuptake
inhibitor (SNRI) and consequently elevate the local concentration of
these two potential neurotransmitters in the descending pain pathways
of brain and the spinal cord; damping pain transmission from the periphery to the CNS and thus relieving pain [22–24]. Duloxetine can be
used also in management of depression, chronic musculoskeletal disorders as fibromyalgia and treatment of allodynia related inflammatory
pain [24,25]. Extensive studies evaluated the role of duloxetine in
managing PDN, either in comparison to placebo or to other drugs like
pregabalin and amitriptyline [24,26–30] or in combination with other
medications such as celecoxib [31], with diverse results. Experimental
studies highlighted the role of duloxetine in alleviating symptom of
PDN [32] and suppressing spinal microglia in a model of chemotherapy-induced neuropathy [33].
The current study aimed to investigate the crucial role of duloxetine
as a sciatic and spinal neuroprotective agent in a mouse model of diabetic neuropathy. As spinal microglia and astrocytes had been shown to
be involved in the pathogenesis of PDN, this study examined for the
first time the effect of duloxetine on spinal expression of microglia and
astrocytes in diabetic animals as a possible mechanism involved in its
curative effect.
2.4.2. Experimental groups
Nine weeks after confirming the development of hyperglycemia,
diabetic mice were tested by von-Frey filaments and hot-plate test to
confirm the development of symptoms of PDN. Diabetic mice with
preliminary tactile allodynia and thermal hyperalgesia were randomly
distributed to the study groups.
The experiment was done on four groups of mice, eight mice each.
Groups were assigned as: saline control group, diabetic control group,
diabetic + duloxetine (15 mg/kg, p.o.) groups and diabetic + duloxetine (30 mg/kg, p.o.) group. In general, oral duloxetine therapy was
given every day at 10 a.m. by oral gavage and was initiated at the beginning of week 10 and continued for four weeks (the end of week 13).
2.4.3. Pain tests
Pain tests were performed at the end of the therapeutic period
(4 weeks) to detect mechanical allodynia and thermal hyperalgesia. The
therapeutic period was chosen according to Muari et al. (2014) who
tested duloxetine for four weeks in alleviating painful peripheral neuropathy via modulating glia and NGF expression [37].
2. Materials and methods
2.4.3.1. Von-Frey filaments. It is employed to measure the response to
innocuous mechanical stimulus to a series of von-Frey filaments (0.16,
0.6, 1.4, 4, 10, 60, 100, 180 and 300 g) in ascending forces using the up
and down method. Each filament was tested by pressing it
perpendicular to the median plantar surface of the right hind paw for
5 times per paw and the mechanical threshold was defined as “the
minimal force that caused at least 3 withdrawals observed out of 5
consecutive trials”. Positive responses were considered with prolonged
withdrawal, licking or biting of the hind paw [38].
2.1. Animals
Fifty-six male Swiss mice (22 ± 4 g) were purchased from Mousfata
Rashed Company for experimental animals (Giza, Egypt). Mice acclimatized to the experimental conditions for one week before starting the
experiment. Mice were placed in polyethylene cages in controlled hygienic conditions and normal light/dark cycle with water and regular
diet given ad libitum. The experimental protocol was approved by the
institutional research ethics committee (license number 201603A6).
2.4.3.2. Hot-plate test. It is employed to measure the response latencies
to an acute noxious thermal stimulus. Each mouse was placed on the
metal plate heated to 55 °C (Lsi LETI- CA, model LE 7406, Italy) and
covered with a glass cylinder (25-cm high, 20-cm in diameter) and the
pharmacological activity of duloxetine was estimated by measuring the
latency period preceding the animal reaction of licking its hind paw or
jump [39]. Cut-off time of 45 s was set in order to prevent tissue
damage [40].
2.2. Drugs and chemicals
Alloxan hydrate was purchased from s d fine-chem limited
(Mumbai, India) as white powder and dissolved in normal saline.
Cymbalta hard gelatin capsules (Eli Lilly Pharmaceutical Company)
containing duloxetine were purchased from the market. The content of
the capsules was suspended in distilled water and given orally to mice.
2.3. Experiment I: assessment of possible serotonin syndrome due to
duloxetine doses
2.4.4. Sacrification of mice and dissection of organs
One day after assessing behavioral tests, the final fasting blood
glucose level was measured. Then, mice were sacrificed by cervical
dislocation and the vertebral column and the sciatic nerves from both
hind limbs were dissected. Right sciatic nerves were kept in RNAlater
stabilizing reagent (Applied BioSystems) at −80 °C for subsequent
evaluation of NGF gene expression while the left sciatic nerves were
kept in phosphate-buffered formalin and used in various histopathological and immunohistochemical studies.
Three groups of mice (8 mice each) were challenged by either distilled water or duloxetine (15 or 30 mg/kg) by an oral gavage every day
for a period of four-weeks to exclude any possible behavioral changes
occurred due to stimulation of the serotonergic system or brain serotonergic activity. Fifteen minutes after water or drug administration,
postures and behaviors associated with the rodent serotonin syndrome
were recorded for five 1-min periods every 5 min [34]. Intermittent
behaviors were evaluated according to backward gait, tics, tremor and
hunched back and scored as: 0, absent; 1, expressed once; 2, expressed
several times; 3, permanently expressed. Additionally, continuous behaviors including flat body position, piloerection, straub tail and hind
leg abduction were observed and a value of 1 is assigned each time that
they were present. A global score was calculated for each mouse by
adding each of the five 1-min periods for each sign [35].
2.4.5. Nerve growth factor gene expression by RT-PCR
The sciatic nerve was isolated and kept in RNAlater stabilizing reagent [Qiagen, cat no 76104] at −20 °C until processed. Isolation of
total RNA was performed using RNeasy FFPE kit [Qiagen, cat no
73504] according to the instructions of the manufacturer. The purity
and concentration of RNA in samples were determined using a
NanoDrop ND-1000 spectrophotometer [NanoDrop Tech., Inc.
Wilmington, DE, USA]. Production of complementary DNA (cDNA) was
done by 10 ng RNA and high capacity cDNA reverse transcription kit
from Applied Biosystems [P/N 4368814] in a mastercycler gradient
thermocycler [Eppendorf, Hamburg, Germany] as previously described
[41]. Real-time polymerase chain reaction (qRT-PCR) was used to
2.4. Experiment II: in vivo neuroprotective activity of duloxetine
2.4.1. Induction of type 1 diabetes mellitus in mice
Mice were fasted overnight then received a single injection of alloxan (180 mg/kg, i.p.) [36]. After one-week, fasting blood glucose was
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determine gene expression profile of NGF (Assay ID:
Mm00443039_m1). The assay reactions were performed in quadruplicate with optimum controls. A 20-μl reaction volume included
10 μl TaqMan® universal PCR master mix [Applied Biosystems, P/N
4440043], 1 μl 1× TaqMan® assay (Applied Biosystems, assay ID
Mm00443039_m1 and Mm99999915_g1 for the endogenous control
GAPDH gene), 1.5 μl cDNA and 7.5 μl of nuclease free water. The PCR
was performed on StepOnePlus Real Time-PCR system (Applied BioSystems) according to the following procedures: 95 °C for a 10-min
period followed by 40 cycles of 95 °C for a 15-s period and 60 °C for
1 min. Relative quantification of the gene in each sample was estimated
using LIVAK method as described before [41].
2.4.6. Histopathological examination of the spinal cord and sciatic nerve
Cross sections from the cervical part of the spinal cord are commonly used for investigating the pathologic features of spinal cord in
neuropathy models [42,43]. One clinical study demonstrated a significant reduction in cross-sectional area of the cervical spine using
magnetic resonance imaging in subjects with advanced diabetic peripheral neuropathy compared with nondiabetic control subjects [44].
Therefore, cervical sections from the spinal cord and sections were
taken from the sciatic nerve were cut and processed for staining with
hematoxylin and eosin (H&E). The spinal cord was scored according to
the presence of eosinophilic foci of degenerated neurons and the level
of gliosis in both the gray and white matter into the following degrees
described previously with some modification: grade (0): no change;
grade (1): +; grade (2): ++; grade (3): +++, grade (4): ++++,
grade (5): +++++. Additionally, the severity of sciatic nerve fiber
degeneration, myelinopathy and axonopathy were classified into the
following degrees: grade (0): no change; grade (1): mild; grade (2):
moderate; grade (3): severe [43].
Fig. 1. Effect of duloxetine on the threshold of withdrawal of animal paws in
von-Frey filaments test. Filaments were applied in an ascending order using the
up and down method. Results are mean ± SEM and analyzed using one-way
ANOVA followed by Tukey's post-hoc test at P < 0.05. aCompared to saline
group, bcompared to diabetic group, ccompared to diabetic+duloxetine
(15 mg/kg) group, n = 6–8.
2.4.7. Immunohistochemistry for GFAP, CD11b and NGF
Cervical sections of spinal cords were dissected and embedded in
paraffin. Then, specimens were cut over a glass slide and subjected to
antigen retrieval. After that, tissue sections were incubated with the
primary antibodies in a humidity chamber according to the manufacturer's instructions. Rabbit polyclonal antibodies against mice GFAP
(Thermo Fisher Scientific, UK, Cat. # RB-087-A) and CD11b (Biorbyt,
UK, Cat. # orb11009) were used. After that, the reaction was visualized
using a Power-StainTM 1.0 Poly HRP DAB kit (Genemed
Biotechnologies, South San Francisco, CA 940800, USA). Similarly,
sciatic specimens were immunostained using rabbit monoclonal NGF
beta antibody [EP1320Y], C-term (GeneTex Inc., Cat. # GTX61496).
Methods were applied according to the manufacturer's instructions.
Then, slides were examined and imaged under a light microscope and
percent of immunopositive areas were determined using an image
analysis system [ImageJ 1.45 F] (National Institute of Health, USA).
30 mg/kg) compared to diabetic control group (426.17 ± 17.6 and
416 ± 38.4 vs. 404.67 ± 30.1). The mortality % in each group was
registered at the end of the experiment, it was 0% in the saline group,
25% in diabetic control group, 12.5% in diabetic + duloxetine (15 mg/
kg) group and 25% in diabetic + duloxetine (30 mg/kg) group (data
not shown in illustrations).
In addition, mice used in the current study were observed for possible development of serotonin syndrome before performing any of the
behavioral tests. However, none of mice groups showed the symptoms
of serotonergic stimulation and therefore, all groups got score “zero”
(data not shown in illustrations).
The current results demonstrated that diabetic mice showed lower
withdrawal threshold when tested using von-Frey filaments compared
to the saline group (3.21 ± 026 vs. 6.74 ± 0.22, Fig. 1). Treatment
with the lower dose of duloxetine (15 mg/kg) did not produce a significant change in the withdrawal threshold. However, treating diabetic
mice with the higher dose of duloxetine (30 mg/kg) increased the
withdrawal threshold compared to the diabetic mice as well as diabetic
mice treated with duloxetine (15 mg/kg) (P < 0.05, Fig. 1).
Results of testing thermal hyperalgesia using the hot-plate test indicated shorter licking time and shorter latency to jumping recorded for
diabetic mice in comparison to the saline treated mice. Treatment of
diabetic mice with duloxetine (15 or 30 mg/kg) prolonged the licking
time and the latency to jumping from the hot-plate test compared to the
diabetic control mice (P < 0.05). The effect of the high dose was not
significantly different from that produced by the low dose of duloxetine
(Fig. 2).
The histopathological examination of specimens from the cervical
part of the spinal cord of albino mice revealed that the saline group
showed normal architecture of the spinal cord which is characterized by
the butterfly contour and consists of central canal, white matter at the
periphery that consists of mainly nerve fibers where the dissolved
myelin sheath left empty spaces that surround dark stain spots (axon).
2.5. Analysis of data
The presentation of the quantitative data was done as mean ±
standard error of the mean. Statistical analysis was performed using the
one-way ANOVA followed by Tukey's post-hoc test. The level of significance was set at P < 0.05. Histologic scores for spinal cords and
sciatic nerves were demonstrated in box-plots representing the median
and quartiles and analyzed by non-parametric ANOVA followed by
Mann-Whitney U test at P < 0.05. Every possible comparison between
the study groups was recorded and showed in illustrations.
3. Results
Measuring fasting blood glucose highlighted significant hyperglycemia in alloxan treated group compared to saline group
(404.67 ± 30.1 vs. 96 ± 4.04, P < 0.05). No change in blood glucose
was observed after treatment with any of duloxetine doses (15 or
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spinal cord specimens revealed that, the diabetic group showed higher
immunoreactivity for GFAP (4.6-fold increase) and CD11b (approximately 2-fold increase) if compared to saline group. On the other hand,
treatment with duloxetine (15 and 30 mg/kg) showed significant reduction (P < 0.05) in the immunoreactivity for GFAP, and only the
high dose reduced CD11b compared to the diabetic group (Figs. 4A&B
and 5A&B).
Histopathological examination of specimens from the sciatic nerve
of albino mice revealed that, the saline group showed normal architecture of the sciatic nerve with regular arrangement of the axons into
fascicles, each fascicle surrounded by connective tissue perineurium.
The nerve bundle contains different size of myelinated nerve fibers
separated by connective tissue endoneurium with its component endoneurial cells. The individual nerve fibers consist of central axon
which appears as dot in the center of the cross section and as wavy
eosinophilic lines in the longitudinal sections, surrounded by empty
space of lipid rich myelin sheath with crescent-shaped nuclei of
Schwann cells. There were scattered thin wall blood vessels in-between
the nerve fibers (Fig. 6Aa&b). However, in the diabetic group, there
were thickening in the perineurium, sub-perineurial edema, vacuolar
degeneration in the endoneurium with less number of endoneurial cells,
thickening and congestion in the blood vessels. Edema and swelling in
some myelin sheath, focal area of hyaline degeneration in the nerve
fiber with aggregation of enlarged Schwann cells and endoneurial cells.
Loss of some axons (Fig. 6Ac&d). On the other hand, moderate improvement was observed in the diabetic + duloxetine (15 mg/kg)
group, as axons were regularly arranged. Examination detected thickening in the perineurium but moderate sub-perineurial edema. Further,
vacuolar degeneration in the endoneurium, residual edema and swelling in the myelin sheath and slight enlargement of Schwann cells were
also appreciated (Fig. 6Ae). Furthermore, there was a significant improvement in the pathological changes in diabetic mice treated with
duloxetine (30 mg/kg) compared to those treated with the low dose of
duloxetine (15 mg/kg). Edema and swelling were observed in the
myelin sheath and some Schwann cells were still enlarged and showing
loss of some axons. Perineurium and endoneurium were relatively
normal (Fig. 6Af). Furthermore, in duloxetine-treated groups, significant decreases in the histopathological score of the degeneration,
myelinopathy and axonopathy were detected compared to the diabetic
group (P < 0.05, Fig. 6B).
The immunohistochemical staining for NGF in the sciatic nerve revealed that the diabetic group showed lower immunoreactivity for NGF
(approximately one half) if compared to saline group. On the other
hand, treatment with duloxetine (15 and 30 mg/kg) produced significant increases in the immunoreactivity for NGF compared to the
diabetic group (Fig. 7A&B).
Fig. 8 demonstrates the PCR determination of sciatic expression for
NGF. Diabetic mice showed lower expression for NGF compared to the
saline group. Treatment with both doses of duloxetine (15 or 30 mg/kg)
increased sciatic expression of NGF compared to the diabetic control
group (P < 0.05, Fig. 8).
Fig. 2. Effect of duloxetine on the licking time and latency time for mice tested
in the hotplate test. A) licking time is the time in seconds till the mouse licks its
forepaws and B) latency time is the time until the animal jumps out of the
apparatus with cut-off time equal 45 s. Temperature was adjusted at 55 °C.
Results are mean ± SEM and analyzed using one-way ANOVA followed by
Tukey's post-hoc test at P < 0.05. aCompared to saline group, bcompared to
diabetic group, n = 6–8.
The gray mater toward the center is predominated with neuronal cell
bodies, astrocytes, microglia cells and abundant cell processes (Fig. 3Aa
&b). However, in the diabetic group, showed gliosis in the gray and
white matter because of increased astrocytes and microglia cells specially the astrocytes (Fig. 4Ab), neuronal necrobiosis which revealed as
pyknotic nucleus, with multiple numbers of homogenous eosinophilic
degenerated neurons, preneuronal edema and status spongiosis in some
areas of the white matter (Fig. 3Ac&d). On the other hand, some improvements were detected in the diabetic + duloxetine (15 mg/kg)
group, but glial nodule and mild gliosis around little degenerated
neurons were still present (Fig. 3Ae). Furthermore, nearly normal
structure of the spinal cord with neuronal cell bodies, astrocytes and
microglia cells with mild gliosis in gray and white matter were detected
as ameliorating effect after treatment with duloxetine (30 mg/kg)
(Fig. 3Af). Furthermore, in the treated groups a significant decrease of
the histopathological score of the neuronal necrobiosis and the level of
gliosis in both the gray and white matter were shown compared to the
diabetic group (P < 0.05, Fig. 3B).
The immunohistochemical staining for GFAP and CD11b in the
4. Discussion
Neuropathic pain had been defined as a complex, chronic pain resulting from the presence of injury or disease in the tissues of the somatosensory system [45]. Metabolic disturbance is the cornerstone in
the pathogenesis of PDN. Activation of the polyol pathway triggered by
the hyperglycemic state is responsible for the nerve damage due to the
increased affinity of aldose reductase to glucose with subsequent formation and accumulation of sorbitol intracellularly resulting in high
osmotic pressure and increased water influx yielding Schwann cell
damage and degeneration of nerve fibers. In addition, upregulation of
NADPH oxidase and increased concentration of reactive oxygen species
(ROS) are known to be involved in developing hypoxia with subsequent
damage in blood vessels which supply the peripheral nerves [46].
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Fig. 3. Histopathological pictures for cervical
sections from the spinal cord stained with hematoxylin and eosin. A) Photomicrographs of
cervical sections from the spinal cord of diabetic
mice. Images represent sections stained with
hematoxylin and eosin (a & b): sections from
saline group showing: normal structure of the
spinal cord represented by, white matter (W),
gray matter (G), central canal (C), neuronal cell
bodies (N), astrocyte (blue arrow) and microglia
cell (red arrow) with abundant cell processes
(head arrow). (c & d): sections from diabetic
group showing, gliosis (blue arrow) in gray and
white mater, neuronal necrobiosis (yellow
arrow) with multiple numbers of homogenous
eosinophilic degenerated neurons (black arrow)
in gray mater, preneuronal edema (E) and status
spongiosis (s). (e): section from diabetic + duloxetine (15 mg/kg) group showing:
glial nodule (GN), mild gliosis (blue arrow)
around degenerated neurons (N). (f): section
from diabetic+duloxetine (30 mg/kg) group
showing: nearly normal structure of the spinal
cord with mild gliosis (blue arrow). Scale
bar = 100 μm for (a) and 50 μm for b–f. B)
Histologic score for the spinal sections: as grade
(0): no change; grade (1): +; grade (2): ++;
grade (3): +++, grade (4): ++++, grade
(5): +++++. Data are box-plots representing
median and quartiles and analyzed by nonparametric ANOVA followed by Mann-Whitney
U test at P < 0.05. aCompared to saline group,
b
compared to diabetic group, ccompared to
diabetic + duloxetine
(15 mg/kg)
group,
n = 6–8. (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this article.)
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(caption on next page)
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Fig. 4. Cervical sections from the spinal cord immunostained for glia fibrillary acidic protein. A) Photomicrographs of cervical sections from the spinal cord
representing the intensity of glia fibrillary acidic protein. Image for saline group (a), diabetic group (b), diabetic + duloxetine (15 mg/kg) group (c) and diabetic + duloxetine (30 mg/kg) group (d), scale bar = 20 μm. B) Area for GFAP immunostaining represented as mean ± SEM. GFAP: glia fibrillary acidic protein.
Data were analyzed using one-way ANOVA followed by Tukey's post-hoc test at P < 0.05. aCompared to saline group, bcompared to diabetic group, n = 6–8.
Fig. 5. Cervical sections from the spinal cord immunostained for CD11b. A) Photomicrographs for cervical sections representing the staining of CD11b. Images for
saline group (a), diabetic group (b), diabetic + duloxetine (15 mg/kg) group (c) and diabetic + duloxetine (30 mg/kg) group (d), scale bar = 20 μm. B) Area for
CD11b immunostaining represented as mean ± SEM and analyzed using one-way ANOVA followed by Tukey's post-hoc test at P < 0.05. aCompared to saline group,
b
compared to diabetic group, n = 6–8.
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Fig. 6. Histopathological picture for sections
from the sciatic nerve stained with hematoxylin
and eosin. A) Photomicrographs for sciatic nerve
in the experimental groups. Images represent
sections stained with hematoxylin and eosin (a
& b): represent longitudinal sections and cross
sections in saline group showing, connective
tissue perineurium (P), endoneurium (curved
arrow), axon of the individual nerve fiber (blue
arrow), surrounded by empty space myelin
sheath (tailed arrow), crescent-shaped nuclei of
Schwann cells (red arrow), node of Ranvier
(yellow arrow), some nuclei of endoneurial cells
(black arrow) and thin wall blood vessels (head
arrow). (c & d): Longitudinal and cross sections
in diabetic group showing, thickening in the
perineurium (P), sub-perineurial edema (E),
vacuolar degeneration in the endoneurium
(curved arrow), thick congested blood vessels
(head arrow). Edema and swelling in the myelin
sheath (tailed arrow), focal area of hyaline degeneration in the nerve fiber (D), enlarged
Schwann cells (red arrow) and loss of some
axons (wavy arrow). (e): Longitudinal section in
diabetic + duloxetine
(15 mg/kg)
group
showing, thickening in the perineurium (P) subperineurial edema (E), vacuolar degeneration in
the endoneurium (curved arrow), residual
Edema and swelling in the myelin sheath (tailed
arrow), and slight enlarged Schwann cells (red
arrow). (f): Longitudinal section in diabetic + duloxetine (30 mg/kg) group showing,
edema and swelling in the myelin sheath (tailed
arrow), slight enlarged Schwann cells (red
arrow) and loss of some axons (wavy arrow),
scale bar = 30 μm for a, c, e and f and =20 μm
for b & d. B) Data from scoring of the sciatic
nerve demonstrated as box-plot representing the
median and quartiles and analyzed using nonparametric ANOVA followed by Man-Whitney U
test. aCompared to saline group, bcompared to
diabetic group, ccompared to diabetic + duloxetine (15 mg/kg) group, n = 6–8. (For interpretation of the references to colour in this
figure legend, the reader is referred to the web
version of this article.)
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Fig. 7. Histopathological sections from the sciatic nerve immunostained for nerve growth factor. A) Photomicrographs for the sciatic nerve representing the intensity
of NGF expression. Images from saline group (a), diabetic group (b), diabetic + duloxetine (15 mg/kg) group (c) and diabetic + duloxetine (30 mg/kg) group (d),
scale bar = 20 μm. NGF: nerve growth factor. B) Area for NGF immunostaining represented as mean ± SEM and analyzed using one-way ANOVA followed by
Tukey's post-hoc test at P < 0.05. aCompared to saline group, bcompared to diabetic group, n = 6–8.
dose of duloxetine (30 mg) in diabetic mice. Similar findings have been
documented by Mixcoatl-Zecuatl and Jolivalt [32]; authors stated that
the intrathecal injection of duloxetine (20 μg) or intraperitoneal injection (20 mg/kg) in diabetic rats decreased the tactile allodynia. In the
present study, it was obvious that duloxetine administration revealed
In the current study, alloxan model was used for induction of DM.
Von-Frey filaments and hot plate tests were used to assess the presence
of mechanical allodynia and thermal hyperalgesia respectively as a
strong evidence of the development of PDN in diabetic mice.
The current study highlighted an antiallodynic effect for the high
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hyperalgesia associated with PDN. Rats treated with lysosomal cysteine
protease cathepsin S (Cat S) inhibitor showed a greater decrease in
spinal microglial activation and pain hypersensitivity as the activated
spinal microglia express Cat S, which contributes in the development of
hyperalgesia [65]. In agreement, it has been documented that targeting
microglial activation inhibits the actions of chemokines, ATP receptors,
and/or proinflammatory cytokines and might lead to novel therapies
for chronic pain [66]. An evidence was provided by Pabreja et al. [68],
who revealed that activated microglia may be involved in the development of PDN and minocycline (a selective inhibitor of microglial
activation) exerted its protective effect by inhibition of the neuroimmune activation of microglia [67].
In the present study, duloxetine blocked spinal microglial and astrocyte activation in PDN. Mice treated with the high dose of duloxetine
(30 mg/kg) showed favorable improvements in the structure of the
spinal cord and decreased gliosis. Similarly, it was previously mentioned that the analgesic effect of duloxetine, in a rat model of intervertebral disc-related neuropathic pain, was linked to suppression of
microglia activation. Taking into consideration that duloxetine, and
most likely other SNRIs, can modulate neuroinflammation by their interaction with serotonin and noradrenaline receptors on microglia [68].
Importantly, P2X4R is a subtype of ATP-gated non-selective cation
channels that is highly upregulated in microglia in PDN. Recently,
Yamashita and his colleagues in 2016, have outlined the inhibitory
effect of duloxetine on recombinant P2X4R (rat and human) and microglial P2X4R (mouse and rat) [69].
Further, the neuroprotective effect of duloxetine in diabetic mice
was indicated previously by improving the sciatic nerve histopathology
[70]. However, one clinical study denied any neuroprotective role for
duloxetine in preventing the development of neuropathy in diabetic
patients [71].
Nerve growth factor is a neurotrophic factor required for the survival of the rat sensory neurons [72]. Its level is dependent on the
proinflammatory mediators released in response to inflammation or
tissue damage. Ramesh et al., 2013 stated that neuroinflammation leads
to nerve damage due to apoptosis induced by MAPK signaling. The
altered pathways [advanced glycation end-product, polyol, hexosamine, protein kinase C and mitogen-activated protein kinases] observed in the pathogenesis of diabetic neuropathy, lead to devastating
changes at the level of gene transcription as well the protein function
with consequent impairment of neurotrophism, axonal transport and
gene expression and ultimately promote the development of diabetic
complications [73]. Low level of NGF was observed in peripheral nerves
in a diabetic rat model; this was linked to a defect in its axonal transport
[74]. Moreover, DM is characterized by degeneration of peripheral
neuron/fibers and altered local levels of NGF/NGF receptors and deregulation of NGF signal pathway [75]. Moreover, in streptozotocindiabetic rats, NGF levels decrease in sympathetically innervated target
organs, the superior cervical ganglion and sciatic nerve [76].
Regarding the sciatic nerve expression of NGF, diabetic mice
showed lower expression in comparison to saline group. Mice treated
with both doses of duloxetine (15 or 30 mg/kg) showed an over expression of NGF. This highlights for the first time the sciatic neurogenerative effect of duloxetine includes upregulation of sciatic NGF
level. Similarly, experimentally diabetic mice showed low levels of NGF
and platelet derived growth factor C (PDGF-C) proteins in the sciatic
nerve tissue compared to non-diabetic mice [77].
In experimental models of diabetic neuropathies, NGF administration reversed the neurodegenerative signs and normalized the activity
of neurons belonging to the peripheral nervous system [78]. Hence,
downregulation of NGF is considered to be one of the most important
factors contributing to the pathogenesis of diabetic neuropathy beside
the chronic hyperglycemia and hypoxia [79,80].
In brief, hyperglycemia promotes neuroinflammation caused by
ROS generation, which in turn causes microglial and astrocytic activation with subsequent nerve damage, apoptosis and defect in axonal
Fig. 8. Effect of duloxetine on the mRNA expression of nerve growth factor.
Results are mean ± SEM and analyzed using one-way ANOVA followed by
Tukey's post-hoc test at P < 0.05. aCompared to saline group, bcompared to
diabetic group, n = 6–8.
effectiveness in attenuating the thermal hyperalgesia in the current
diabetic mouse model. Similarly, a dose-dependent anti-nociceptive
effect of duloxetine (5, 10 and 20 mg/kg, i.p.) was highlighted in tailimmersion and hot-plate tests in diabetic mice [46]; this confirms the
results obtained previously from clinical studies on diabetic patients
[47–49]. Such effects exerted by duloxetine are thought to be attributed
to suppressing serotonin and noradrenaline transporters; leading to
increased levels and persistent actions of these neurotransmitters in the
descending inhibitory pathways [50]. Furthermore, multiple studies
demonstrated the effective role of duloxetine as an anti-nociceptive
drug in injured and inflammatory models [24,51–55].
In the current study, the histopathological examination of the spinal
cord of the diabetic group showed extensive gliosis caused by the robust
increase of microglial and astrocytic cells. Similar results were reported
previously confirming the role of microglial and astrocytic activation in
maintenance of painful episodes in streptozotocin-induced PDN in rats
[56,57]. Following microglial activation, which is evidenced by the
proliferation of microglia in the spinal cord, microglia change in morphology, express new surface cell markers (as CD11b and CD14), migrate
to the site of injury and secrete several proinflammatory cytokines;
contributing to the phagocytic process and consequently provoking
allodynia, hyperalgesia and nociception [58].
While microglia attributed to the development of pain at earlier
stages, pain sensation is sustained by astrocytes. Garrison et al. (1991)
firstly described the presence of activated astrocytes in the diseased
spinal cord after sciatic nerve injury in rat model [59]. Astrocytes activation begins relatively later and increased much slower in comparison to microglial activation [60–62]; causing further microglial activation in an amplifying cascade [63]. Astrocytic changes include the
release of different cytokines in addition to the overexpression of GFAP,
a major astrocytic marker which in turn play a crucial role in development of hyperalgesia and chronic pain [64].
Blockage of spinal microglial and astrocytic activation is an encouraging goal in the treatment of neuropathic pain, allodynia and
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M.K. Tawfik et al.
transport causing sharp decline in the levels of NGF seen in PDN
[73,74]. On the other hand, microglia inactivation produced by duloxetine is expected to suppress the inflammatory reactions involved in
PDN after peripheral nerve injury and restore peripheral nerve function
[69] as well as neuroregeneration with subsequent elevation of NGF.
[15]
[16]
[17]
5. Conclusion
Microglial inactivation produced by duloxetine contributed to protection against peripheral nerve injury and helped for restoring peripheral nerve structure with elevation of sciatic NGF. Duloxetine improved pain-related behavior in the current mouse model of PDN. This
ameliorating effect was thought to be, at least partly, mediated by
suppression of spinal glia expression and restoring sciatic integrity via
upregulating NGF expression. As duloxetine is currently reported safe
for diabetic patients, it may be sound to call for more research projects
to explain the exact mechanism by which duloxetine inhibits microglial
activation in PDN. Additionally, further studies are warranted to fully
elucidate the neuroprotective role of other SNRI in PDN.
[18]
[19]
[20]
[21]
Funding sources
[22]
Self.
[23]
Conflicts of interests
None.
[24]
Acknowledgments
[25]
Authors thank Pathologist/Ahmed Abd-Allah, Faculty of Medicine,
Ain Shams University for help in the dissection of the sciatic nerves.
[26]
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