Life Sciences 205 (2018) 113–124 Contents lists available at ScienceDirect 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 . 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  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 . Astrocytes are the most abundant glial cells representing 40%–50% of CNS glial cells . Moreover, microglia had been recently involved in the pathogenesis of neuropathic pain since they become activated upon injury . 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) . 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 . 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 , antiepileptics including pregabalin or opioids (nonspeciﬁc analgesics) . 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 . 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 , with diverse results. Experimental studies highlighted the role of duloxetine in alleviating symptom of PDN  and suppressing spinal microglia in a model of chemotherapy-induced neuropathy . 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 . 2. Materials and methods 188.8.131.52. 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 . 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). 184.108.40.206. 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 . Cut-oﬀ time of 45 s was set in order to prevent tissue damage . 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 . 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 . 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 . 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.) . After one-week, fasting blood glucose was 114 Life Sciences 205 (2018) 113–124 M.K. Tawﬁk et al. 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 . 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 . 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 . 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 115 Life Sciences 205 (2018) 113–124 M.K. Tawﬁk et al. 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 . 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 . 116 Life Sciences 205 (2018) 113–124 M.K. Tawﬁk et al. 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.) 117 Life Sciences 205 (2018) 113–124 M.K. Tawﬁk et al. (caption on next page) 118 Life Sciences 205 (2018) 113–124 M.K. Tawﬁk et al. 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. 119 Life Sciences 205 (2018) 113–124 M.K. Tawﬁk et al. 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.) 120 Life Sciences 205 (2018) 113–124 M.K. Tawﬁk et al. 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 ; 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 121 Life Sciences 205 (2018) 113–124 M.K. Tawﬁk et al. 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 . 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 . An evidence was provided by Pabreja et al. , 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 . 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 . 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) . Further, the neuroprotective eﬀect of duloxetine in diabetic mice was indicated previously by improving the sciatic nerve histopathology . However, one clinical study denied any neuroprotective role for duloxetine in preventing the development of neuropathy in diabetic patients . Nerve growth factor is a neurotrophic factor required for the survival of the rat sensory neurons . 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 . Low level of NGF was observed in peripheral nerves in a diabetic rat model; this was linked to a defect in its axonal transport . Moreover, DM is characterized by degeneration of peripheral neuron/ﬁbers and altered local levels of NGF/NGF receptors and deregulation of NGF signal pathway . Moreover, in streptozotocindiabetic rats, NGF levels decrease in sympathetically innervated target organs, the superior cervical ganglion and sciatic nerve . 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 . In experimental models of diabetic neuropathies, NGF administration reversed the neurodegenerative signs and normalized the activity of neurons belonging to the peripheral nervous system . 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 ; 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 . 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 . 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 . Astrocytes activation begins relatively later and increased much slower in comparison to microglial activation [60–62]; causing further microglial activation in an amplifying cascade . 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 . Blockage of spinal microglial and astrocytic activation is an encouraging goal in the treatment of neuropathic pain, allodynia and 122 Life Sciences 205 (2018) 113–124 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  as well as neuroregeneration with subsequent elevation of NGF.    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.     Funding sources  Self.  Conﬂicts of interests None.  Acknowledgments  Authors thank Pathologist/Ahmed Abd-Allah, Faculty of Medicine, Ain Shams University for help in the dissection of the sciatic nerves.  References  N.G. Forouhi, N.J. Wareham, Epidemiology of diabetes, Medicine (Baltimore) 42 (12) (2014 Dec 1) 698–702, http://dx.doi.org/10.1016/j.mpmed.2014.09.007.  R. Pop-Busui, A.J.M. Boulton, E.L. Feldman, V. Bril, R. 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