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Neuron-astrocyte interactions in spinal cord dorsal horn in neuropathic pain development and docosahexaenoic acid therapy
Journal of Neuroimmunology 298 (2016) 90–97
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Neuron-astrocyte interactions in spinal cord dorsal horn in neuropathic pain development and docosahexaenoic acid therapy Igor V. Manzhulo a,b,⁎, Olga S. Ogurtsova a, Yuliya O. Kipryushina a, Nikolay A. Latyshev a, Sergey P. Kasyanov a, Inessa V. Dyuizen a, Anna A. Tyrtyshnaia a,b a b
A.V. Zhirmunsky Institute of Marine Biology, Far Eastern Branch, Russian Academy of Sciences, Vladivostok, 17 Palchevskii Str., 690041, Russia School of Biomedicine, Far Eastern Federal University, Vladivostok, 8 Sukhanova Str., 690950, Russia
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Article history: Received 17 March 2016 Received in revised form 15 June 2016 Accepted 15 July 2016 Keywords: Spinal cord dorsal horn Docosahexaenoic acid Chronic constriction injury Astrocyte Substance P Nitric oxide synthase
a b s t r a c t The analgesic activity of docosahexaenoic acid (DHA, 22:6 n−3) was studied using a chronic constriction injury (CCI) rat model. Animals were subcutaneously injected with DHA emulsion at a dose of 4.5 mg/kg (125 mМ/kg) daily during 2 weeks after surgery. We characterized the dynamics of GFAP-positive astrocyte, substance P (SP) and nNOS-positive neurons activity in the spinal cord dorsal horn (SCDH) superficial lamina. We found that DHA treatment decrease the intensity and duration of neurogenic pain syndrome, results in earlier stabilization of weight distribution, prevents the cold allodynia and dystrophic changings in denervated limb tissue. DHA treatment reduced the reactive astrocyte number, decrease SP-immunopositive fibers and nNOS-positive neurons number in the SCDH in neuropathic pain. © 2016 Elsevier B.V. All rights reserved.
1. Introduction To date, pain and its treatment is one of the most topical issue in fundamental and practical neuroscience. Neuropathic pain, caused by primary lesion or dysfunction of somatosensory nervous system (Dworkin et al., 2007), results in development and progression of many somatic and psychophysiological disorders (Costigan et al., 2009). Despite the widespread clinical application of analgesic drugs of different pharmacological groups, achievement of complete pain control is very difficult task, which requires a search for new molecular targets to provide an analgesic effect (Schlereth and Birklein, 2008). Recent data confirm the significant role of glial cells in the pain pathology. Pain signaling is invariably accompanied by structural and functional changes of all glial cells types (Mika et al., 2009). Glial cell properties, including the degree of activation and secreted factors may have a different effect on neuropathic pain severity and duration (Milligan et al., 2003; Milligan and Watkins, 2009). Considering the importance of glial cells and their signaling molecules, development of new therapeutic approaches regulating glial cells activity is a promising direction in pain management.
⁎ Corresponding author at: Laboratory of Pharmacology, A.V. Zhirmunsky Institute of Marine Biology, Far Eastern Branch, Russian Academy of Sciences, Vladivostok, 17 Palchevskii Str., 690041, Russia. E-mail address: [email protected] (I.V. Manzhulo).
http://dx.doi.org/10.1016/j.jneuroim.2016.07.015 0165-5728/© 2016 Elsevier B.V. All rights reserved.
Polyunsaturated fatty acids (PUFAs) demonstrating anti-inflammatory and antioxidant activity are of considerable interest (Farooqui et al., 2007; Tabakaeva and Tabakaev, 2015). Among the most abundant omega-3 PUFAs in nervous tissue, docosahexaenoic acid (DHA) is critically important for nervous cell functional integrity (Bazan, 2007; Nakamoto and Nishinaka, 2010). A deficiency of DHA in brain tissue may affect the activities of membrane-bound enzymes, ion channels, and receptors eventually leading to disruption of neurotransmission and disturbances in brain functioning (Horrocks and Farooqui, 2004). Neuroprotectin D1 (NPD1), the bioactive metabolite of DHA, exerts neuroprotective, anti-inflammatory and anti-apoptotic bioactivity (Serhan, 2006; Xu et al., 2013). We hypothesized that DHA analgesic effect may be related to its influence on neuron-astrocyte interactions in SCDH superficial lamina.
2. Material and methods 2.1. Animals Male Wistar rats (240 ± 20 g, age 2 months, n = 28) were used in experiments. The rats were housed 2 to 4 per cage with ad lib access to food and water. Animals were maintained at a constant temperature (23 ± 2 °C) and humidity (55 ± 15%) with a 12-h light/dark cycle (lights on 7:00 a.m.). The rats were handled for 5 min once a day during 5 days before the experiments. All procedures were approved by the Animal Ethics Committee at the A.V. Zhirmunsky Institute of Marine
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Biology, Far Eastern Branch, Russian Academy of Sciences, according to the Laboratory Animal Welfare guide lines. 2.2. Drugs A high-purified docosahexaenoic acid was produced in Laboratory of Pharmacology, A.V. Zhirmunsky Institute of Marine Biology, Far Eastern Branch, Russian Academy of Sciences. Briefly, docosahexaenoic (DHA, 22:6 n3) acid was extracted from squid Berryteuthis magister liver. The process consisted of three steps: (1) concentration of the total polyunsaturated fatty acids (PUFA) with urea; (2) separation of individual FA by iodolactonization method, and (3) obtaining of highly purified DHA by preparative HPLC. The urea/FA ratio during the first stage of manufacturing process was 3:1, the temperature: 0 and − 20 °C, allowing to increase the DHA concentration from 6.5% to 32.0%. The iodolactonization method was used for separation and purification of individual PUFA. The final concentration of DHA was 95.6%. Stock solution of DHA was prepared by dissolving in 0.9% saline (1:2). Animals were subcutaneously injected with DHA emulsion at a dose of 4.5 mg/kg (125 mМ/kg) (volume of injection was 150 μl) daily during 2 weeks after surgery. As a control drug we used nonsteroidal anti-inflammatory drug diclofenac at a dose of 4 mg/kg. The dose was selected according to the manufacturer's instructions (Indus Pharma, India). 2.3. Surgical procedures A chronic sciatic nerve constriction injury as neuropathic pain model was performed as previously described by Bennett and Xie (1988). Briefly, rats were anesthetized with sodium pentobarbital (50 mg/kg, i.p.), and the left common sciatic nerve was exposed at the middle of the thigh. Three loosely constrictive ligatures (3 silk gut sutures) were tied around the nerve with spacing of approximately 2 mm. Animals were divided into 4 groups (n = 28): “control” group – animals injected with saline (n = 7); a “CCI” group – animals with sciatic nerve constriction injury (n = 7); a “CCI + DHA” group (n = 7) – DHA-injected animals with sciatic nerve constriction injury and a “CCI + diclofenac” group (n = 7) – diclofenac-injected animals with sciatic nerve constriction injury. 2.4. Evaluation of weight bearing deficit Hind paw weight bearing difference was studied as described previously by Nakazato-Imasato and Kurebayashi (2009) using incapacitance tester (Columbus Instruments, USA). This test assesses the severity of pain in the injured limb by determining the weight distribution between the hind paws when animal is fixed in testing chamber. Briefly, the rats were placed into the plexiglass chamber with separate sensor panels for the right and left hind paw. The pressure of each paw exerted on the sensor surface was measured within 3 s. Three measurements with the 5 minute intervals were performed for each animal. The weight distribution for the right and left paws (in grams) was expressed as a percentage of total animal hind paw weight distribution. 2.5. Evaluation of cold allodynia Cold/Hot Plate Analgesia Meter (Columbus Instruments, USA) was used for the evaluation of cold allodynia, always accompanying the development of neurogenic pain syndrome (Bennett, 1993). The test was performed in the chamber with acrylic walls (30 cm high) equipped with metal flor plate (30 × 30 cm) cooled to 0°. Intact animals are able to stand on the cooled floor equally distributing the weight between all paws for a long time. When nerve is damaged, the duration of contact with the cold plate is significantly reduced. To quantify this parameter, we recorded the limb retention time while animal was in the test chamber for 1 min. All tests were performed daily after surgery. Each animal was tested three times with 5 min intervals between measurements.
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2.6. Immunohistochemistry Collection of material for subsequent histological investigation was performed on day 28. Rats were anesthetized with an overdose of sodium thiopental and transcardially perfused with 100 ml of ice cold saline (~4 °C) followed by 100 ml cold fixative (4% paraformaldehyde in 0.1 M phosphate buffer (PBS), pH 7.2). Lumbar segment of the spinal cord was removed immediately and postfixed for 6 h at 4 °C in fresh buffered 4% paraformaldehyde. After rinsing the material was transferred to 20% sucrose for 24 h at 4 °C. Thereafter, the samples were transferred to a Neg 50 (Thermo Scientific, USA) medium for a day and transverse 50 μm sections were obtained with a cryostat microtome (НМ525, Thermo Scientific, USA). Free-floating sections were pre-incubated in 3% hydrogen peroxide to block endogenous peroxidase activity prior to immunohistochemical staining. After three washes in PBS, sections were incubated in blocking buffer containing 2% bovine serum albumin solution (Santa Cruz, SC-2323, USA) and 0.25% Triton X-100 (Gerbu, USA) during 60 min. Sections of spinal cord were incubated overnight at 4 °C with primary antibodies: anti-GFAP mouse monoclonal antibodies, 1:200, Vector Laboratories (VP-G805), USA; anti-substance P mouse monoclonal antibodies, 1:2000, Abcam (ab14184), USA; anti-nNOS rabbit polyclonal antibodies, 1:2000, Abcam (ab40662), USA. Appropriate secondary antibodies conjugated to horseradish peroxidase (PI-1000, anti-rabbit; PI-2000 anti-mouse) were used according to the manufacturer's instructions (Vector Laboratories, USA, 1:100). Sections were incubated in a secondary antibody solution for 45 min at room temperature. After washing, sections were treated for 5– 10 min with chromogen (Nova RED substrate kit, Vector Laboratories, SK-4800, USA) to elicit the immunoperoxidase reaction. The slices were washed with PBS, dehydrated and embedded in Dako TolueneFree Mounting Medium (Dako, CS705, USA). Slides were visualized using light microscopy (Axio Scope A1, Carl Zeiss, Germany), and images were captured using a digital camera (AxioCam ICc3, Carl Zeiss, Germany). 2.7. Image analysis Microphotographs of lumbar spinal cord dorsal horn (SCDH) ipsilateral side were captured and stored as TIFF files. The resulting images were processed and analyzed using ImageJ software (NIH, USA). The number of nNOS-positive neurons in superficial lamina of the SCDH was counted in every eighth slice. The number of nNOS immunopositive cells in the lumbar spinal cord dorsal horn lamina I-III per 1 mm3 of slice was calculated. The number of images for counting was at least 70 per group. The area of GFAP-positive astrocytes and SP-immunopositive fibers in lumbar spinal cord dorsal horn lamina I-III immunohistochemical staining was determined using ImageJ software (NIH, USA) (plugin: IHC Toolbox). 2.8. Statistical analysis The data obtained by immunohistochemistry studies and physiological tests were subjected to statistical analysis using one-way ANOVA tests followed by a post hoc Tukey's multiple comparison test. Repeated measures ANOVA was carried out to evaluation of weight bearing deficit. Data were shown as mean ± SEM and p b 0.05 was taken as statistically significant. All statistical tests were performed using the Microsoft Excel software. 3. Results 3.1. Effects of DHA and diclofenac treatment on behavior in CCI The duration of animals' observation in our study was determined by the active manifestation of test parameters; as we wanted demonstrate active action of DHA in the deferred period after drug
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administration. Control and presurgery animals symmetrically distribute their weight on the hind limbs, and weight distribution symmetry changes dynamically during pain development. At 4–7 days after nerve injury, animals in the “CCI” group distribute the weight as follows: 71.6 ± 0.7% (intact limb) and 28.4 ± 0.7% (injured limb). At the same time period group “CCI + DHA” demonstrated weight distribution between intact and injured limb: 66.9 ± 1%:33.1 ± 1%, group “CCI + diclofenac”: 70.1 ± 2%:29.9 ± 2%. Since day 20 more equal weight distribution between two hind limbs was found in all groups. At 26–28 days weight distribution in animals of “CCI” group for intact and injured limbs was 63 ± 1%:37 ± 1%, while in “CCI + DHA” and “CCI + diclofenac” groups these indexes were 64.1 ± 1%:35.9 ± 1% and 60 ± 1%:40 ± 1%, respectively (Fig. 1A). Cold allodynia testing showed the reduction of the limb contact time in the “CCI” group at 4–7 days postsurgery (10.5 ± 1.9 s). This parameter increased sharply at 8–15 days postsurgery (approximately 24 s) and further trended toward a reduction. In group “CCI + DHA” cold allodynia was not observed. In group “CCI + diclofenac” allodynia
symptoms appeared at 4–7 days and increased to 8–11 days, but we did not observe cold allodynia at the end of our experiment (Fig. 1B). Trophic lesions and autotomy of one or more fingers on the denervated limb were identified in 60% of animals in the “CCI” group (Fig. 2). In groups “CCI + DHA” and “CCI + diclofenac” such pathological changes were not observed. 3.2. Effects of DHA on SCDH superficial lamina astrocytes after CCI Astrogliosis is a typical astrocyte response to peripheral nerve injury accompanying by glial fibrillary acidic protein (GFAP) up-regulation (Ren, 2010; Donegan et al., 2013). Reactive astrocytes release nitric oxide, pro-inflammatory and anti-inflammatory cytokines, matrix metalloproteinases, and other factors that can contribute to neuronal ion homeostasis shifts and altering of metabolic and neurotransmitter properties (Hald et al., 2009). Peripheral nerve injury and the treatment by analgesic drugs are followed by astrocytes number and morphology dynamic alterations. Morphology of GFAP-positive cells in spinal cord
Fig. 1. Time course of DHA and diclofenac analgesic effects on CCI-induced behavior in rats. (A) The dynamics of hind limb weight distribution. (B) The dynamics of cold allodynia (the duration of hind limb lifting above the cold plate for the 1 min observation). Dotted line is “control” group. *Significant differences was observed between groups “CCI” and “CCI + DHA”, “CCI + diclofenac” was at similar observation points (p b 0.05).
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surface plates after sciatic nerve injury is characterized by processes retraction and cell bodies hypertrophy (Fig. 3B). These morphological rearrangements were not typical for animals treated with DHA. In control animals reactive astroglia covers 38.8 ± 3.7% of plate surface area. On day 28 after the sciatic nerve ligation the density of GFAPpositive astrocytes in spinal cord surface plates in groups “CCI” and “CCI + diclofenac” increased to 49.9 ± 1.5% and 49.8 ± 2%, respectively. The difference between “CCI + DHA” and control groups was not significant (Fig. 3 C, D, E). 3.3. Increasing of SCDH superficial lamina SP-immuno-positive staining in CCI-animals after DHA treatment
Fig. 2. Denervated limb of animal in the “CCI” group. White arrow - trophic ulcer, black arrow - autotomy finger.
Substance P is one of the most important mediators of neuropathic inflammatory pain, taking part in the synthesis and release of numerous pro-nociceptive and pro-inflammatory mediators. In the central nervous system SP plays an important role in the spinal cord neurons sensitization, while in the peripheral nervous system, it is involved in the
Fig. 3. Distribution of GFAP-positive astroglia in the spinal cord dorsal horn in animals with neuropathic pain. (A) “control” group; (B) “CCI” group; (C) “CCI + DHA” group; (D) “CCI + diclofenac” group; (E) astroglia activity dynamics. *Significant differences were observed between group “control” and “CCI”, “CCI + diclofenac” at similar observation points (p b 0.05). + Significant differences between groups “CCI” and “CCI + DHA” were at similar observation points (p b 0.05). #Significant differences between groups “CCI + DHA” and “CCI + diclofenac” were at similar observation points (p b 0.05). Scale bar is 200 μm.
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vasodilatation, decreasing of nociceptive activity threshold, as well as in the maintenance of neurogenic inflammation (Maggi, 1995). Thus, modulation of substance P production in the central and peripheral nervous system is suggested to be determinative way of neuropathic pain management and suppression. Accumulations of tightly interwoven SP-positive fibers were observed in the spinal cord surface plates of animals from all experimental groups (Fig. 4). The area of SP-positive fibers in the spinal cord surface plates was 35.9 ± 2% in control animals. SP-immunoreactivity of spinal the cord surface plates in “CCI” and “CCI + diclofenac” animals at day 28 after surgery was increased to 43.36 ± 1% and 42.7 ± 1.9%, respectively (Fig. 4 A, B, D). The area of immunopositive SP-fibers in the group “CCI + DHA” remained at the level of the «control» (Fig. 4 C, E).
Cizkova, 2000) and antinociception (Duarte et al., 1992). Nitric oxide synthesis system is highly dynamic and constantly undergoes quantitative and qualitative changes in pain conditions (Leonard et al., 2001). NO release during the activation of neuronal and inducible NO-synthase underlies the behavioral manifestations of pain such as cold allodynia and thermal hyperalgesia (Onal et al., 2003). In the control group the number of nNOS-positive neurons in the spinal cord surface plates was 2154.5 ± 221.3 per 1 mm3 (Fig. 5 A). Sciatic nerve damage in the “CCI” group was accompanied by the number of neurons expressing NO-synthase increase to 3245.4 ± 166.6 (Fig. 5 B). The use of DHA and diclofenac prevented the increase of nNOS-positive neurons number. Thus, the number of neurons in groups “CCI + DHA” and “CCI + diclofenac” was 1749.3 ± 235.7 and 1962.2 ± 174.4 1 mm3, respectively (Fig. 5 C, D, E).
3.4. nNOS-positive neurons in SCDH superficial lamina in CCI-animals after DHA treatment
4. Discussion
Pharmacological correction of nitric oxide (NO) release significantly influence the nervous system functioning, including neuromodulation (Kiss, 2000), synaptic plasticity (Holscher, 1997), pain (Luo and
Our data demonstrate that CCI in rats is accompanied by cold allodynia and weight distribution asymmetry as well as degenerative changes in denervated limb tissues. These pathologic changes occur at
Fig. 4. Distribution of SP-positive presynaptic sensory fibers in the spinal cord dorsal horn of animals with neuropathic pain. (A) “control” group; (B) “CCI” group; (C) “CCI + DHA” group; (D) “CCI + diclofenac” group; (E) SP activity dynamics. ⁎Significant differences between groups “control” and “CCI” were observed, “CCI + diclofenac” were at similar observation points (p b 0.05). + Significant differences between groups “CCI” and “CCI + DHA” were at similar observation points (p b 0.05). Scale bar is 200 μm.
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Fig. 5. nNOS-positive neurons in the spinal cord dorsal horns of animals with neuropathic pain. (A) “control” group; (B) “CCI” group; (C) “CCI + DHA” group; (D) “CCI + diclofenac” group; (E) nNOS-positive neuron activity dynamics. ⁎Significant differences between groups “control” and “CCI” were at similar observation points (p b 0.05). + Significant differences between groups “CCI” and “CCI + DHA” were observed, “CCI + diclofenac” were at similar observation points (p b 0.05). Scale bar is 200 μm.
day 4 and have been observed for 28 days after surgery gradually decreasing. The absence of cold allodynia 4 weeks after CCI is not an indicator of pain suppression, as other manifestations of pathological sensitivity (tactile allodynia and thermal hyperalgesia, phantom pain) are also present and even become more intense (Malmberg and Basbaum, 1998). Here we found that neuropathic pain development causes cellular changes in the SCDH superficial lamina. On day 28 after CCI modeling we observed astroglial reactivation accompanied by an increase in neurokinin system activity and enhancement of NO-ergic neurotransmission. We assume that SP immunoreactivity increase is related to peripheral sensitization development as well as with the uncontrolled axonal growth at the site of damage, neurotrophic factors production and neuromas formation (Zimmermann, 2001). Our results demonstrate that activation of neurokinin neurotransmission is dynamically coordinated with the astrocytic glia activation in the spinal cord posterior horn. However, the detailed characteristics of SP-ergic system and spinal cord astrocytes interactions are have to be determined. Nevertheless, it is known that astrocytes express surface neurokinin receptors and get direct projections from SP-ergic afferents (Yihong et al.,
2006) and can be activated by neurokinin afferent signaling. In turn, the release of SP by axons and the activity of the neurokinin system in pain may be under the influence of astrocytes - pharmacological inhibition of its activity is accompanied by a decrease in pain through inhibiting of SP synthesis and release (Guo et al., 2007). In this study, the behavioral testing of cold allodynia did not reveal any direct relationship between astrocytic and neurokinin activity in the spinal cord posterior horn and the severity of the pain behavior in the experimental animals. A number of studies demonstrated that the pharmacological regulation of astrocytes activity, as well as inhibition of SP-ergic neurotransmission is capable of tactile allodynia prevention (Nichols et al., 1999; Clark et al., 2007). Thus, the increase of astrocytes and neurokinin system activity at day 28 after surgery may be related to the development of tactile allodynia. Increased NO-ergic neurotransmission is repeatedly described on various experimental models (Meller et al., 1992; Costigan et al., 2009; Keilhoff et al., 2013) and our findings are broadly consistent with the existing experimental and clinical observations. In our study, the administration of DHA is resulted in a stabilization of the earlier weight distribution and prevents the development of cold
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allodynia and dystrophic changes in the denervated limb tissues. Most authors explain this effect by systemic anti-inflammatory and antioxidant activity of DHA (Bazan, 2007; Cunnane et al., 2009). However, the weight distribution at the end of the observation period in group “CCI + DHA” did not differ significantly from group “CCI”, possibly due to termination of drug administration at day 14 after surgery. On day 28 after the surgery, we found no significant differences in such indicators as cold allodynia and weight distribution between the “CCI” and “CCI + DHA” groups. However, at day 28 DHA reduces the level of SP- and NO-ergic neurotransmitter systems activity and astrocytosis in SCDH superficial lamina to the control group level. Some mechanisms responsible for DHA analgesic activity could be suggested. Here, along with suppression of astrocytosis, DHA possibly inhibit the release of neurotrophic factors and inflammatory mediators by astrocytes which are responsible for neural tissue excitability enhancement and neuropathic pain development (Paterniti et al., 2014; Marques et al., 2015). Furthermore, recent studies demonstrate that PUFA, particularly docosahexaenoic acid is an endogenous ligand of PPAR-α (Peroxisome Proliferator Activated Receptor), expressed constitutively on neurons and astrocytes. Thus, among the possible mechanisms of DHA activity, the authors suggest the activation of PPAR-α, which in turn regulates lipid metabolism, glucose homeostasis, as well as demonstrate anti-inflammatory and anti-proliferative properties (Paterniti et al., 2014). Previously it was found that substance P acts as a mediator of neurogenic inflammation by stimulating the cytokines release in damaged tissues (Wei et al., 2012). Docosahexaenoic acid, in addition to the detected neurokinin neurotransmission inhibition may reduce pro-inflammatory cytokines and chemokines expression by the formation of active metabolite neuroprotectin D1 (Lukiw et al., 2005; Xu et al., 2013). According to our previous study, important factors leading to SP-ergic afferent currents stabilization in animals treated with DHA, is a reduction of apoptosis and gliosis in spinal ganglia neurons (Manzhulo et al., 2015) as well as the inhibition of astrocytosis in the spinal cord posterior horns. Previously, we found that DHA reduces DRG microglia/macrophage activity in animals with CCI (Manzhulo et al., 2015). Since the severity of microglial activation is determined by NO-ergic neurotransmission (Kuboyama et al., 2011) the established reduction of NO-ergic neurons number may indicate a direct inter-modulating effects of microglial cells and NO-ergic nociceptive neurons. This is also confirmed by the fact that in mice knockout for each of the three nitric oxide (NO) synthase (NOS) genes the pain reduction is closely related to microglial response suppression in spinal pain centers (Kuboyama et al., 2011). Diclofenac treatment in CCI animals leads to the weight distribution improvement, reduction of cold allodynia and dystrophic changes in the denervated limb tissues. However, it was less effective the DHA treatment. Nevertheless, this study revealed a decreased nNOS activity in SCDH superficial lamina neurons during DHA and diclofenac administration. Based on this, we assume that it is the inhibition of NO-ergic neurotransmission leads to improved physiological parameters in animals with CCI.
5. Conclusions Our study clearly demonstrates that the activation of neurokinin neurotransmission and nNOS synthesis are dynamically coordinated with the astroglial activation in the SCDH superficial lamina during neuropathic pain development. However, the detailed mechanisms of interaction between SP-, NO-ergic systems and astrocytes of the spinal cord remain to be elucidated. Systemic administration of docosahexaenoic acid to CCI animals reduced the intensity and duration of neurogenic pain syndrome, results in earlier stabilization of weight distribution, prevents the cold allodynia and dystrophic changings in denervated limb tissue. DHA treatment reduced the level of the SP- and NO-ergic neurotransmission and decreased astrocytosis in the SCDH superficial lamina. It can be concluded that DHA analgesic activity in neuropathic
pain is related to suppression reactive astrocyte and the inhibition of SP- and NO-signaling pathways.
Acknowledgements Histological and immunohistochemical studies carried out with the Russian Science Foundation financial support (agreement No. 14-5000034), obtaining DHA and all manipulations with animals and image analysis of the material was funded by RFBR according to the research project No. 16-34-00023 mol_a.
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Journal of Neuroimmunology journal homepage: www.elsevier.com/locate/jneuroim
Neuron-astrocyte interactions in spinal cord dorsal horn in neuropathic pain development and docosahexaenoic acid therapy Igor V. Manzhulo a,b,⁎, Olga S. Ogurtsova a, Yuliya O. Kipryushina a, Nikolay A. Latyshev a, Sergey P. Kasyanov a, Inessa V. Dyuizen a, Anna A. Tyrtyshnaia a,b a b
A.V. Zhirmunsky Institute of Marine Biology, Far Eastern Branch, Russian Academy of Sciences, Vladivostok, 17 Palchevskii Str., 690041, Russia School of Biomedicine, Far Eastern Federal University, Vladivostok, 8 Sukhanova Str., 690950, Russia
a r t i c l e
i n f o
Article history: Received 17 March 2016 Received in revised form 15 June 2016 Accepted 15 July 2016 Keywords: Spinal cord dorsal horn Docosahexaenoic acid Chronic constriction injury Astrocyte Substance P Nitric oxide synthase
a b s t r a c t The analgesic activity of docosahexaenoic acid (DHA, 22:6 n−3) was studied using a chronic constriction injury (CCI) rat model. Animals were subcutaneously injected with DHA emulsion at a dose of 4.5 mg/kg (125 mМ/kg) daily during 2 weeks after surgery. We characterized the dynamics of GFAP-positive astrocyte, substance P (SP) and nNOS-positive neurons activity in the spinal cord dorsal horn (SCDH) superficial lamina. We found that DHA treatment decrease the intensity and duration of neurogenic pain syndrome, results in earlier stabilization of weight distribution, prevents the cold allodynia and dystrophic changings in denervated limb tissue. DHA treatment reduced the reactive astrocyte number, decrease SP-immunopositive fibers and nNOS-positive neurons number in the SCDH in neuropathic pain. © 2016 Elsevier B.V. All rights reserved.
1. Introduction To date, pain and its treatment is one of the most topical issue in fundamental and practical neuroscience. Neuropathic pain, caused by primary lesion or dysfunction of somatosensory nervous system (Dworkin et al., 2007), results in development and progression of many somatic and psychophysiological disorders (Costigan et al., 2009). Despite the widespread clinical application of analgesic drugs of different pharmacological groups, achievement of complete pain control is very difficult task, which requires a search for new molecular targets to provide an analgesic effect (Schlereth and Birklein, 2008). Recent data confirm the significant role of glial cells in the pain pathology. Pain signaling is invariably accompanied by structural and functional changes of all glial cells types (Mika et al., 2009). Glial cell properties, including the degree of activation and secreted factors may have a different effect on neuropathic pain severity and duration (Milligan et al., 2003; Milligan and Watkins, 2009). Considering the importance of glial cells and their signaling molecules, development of new therapeutic approaches regulating glial cells activity is a promising direction in pain management.
⁎ Corresponding author at: Laboratory of Pharmacology, A.V. Zhirmunsky Institute of Marine Biology, Far Eastern Branch, Russian Academy of Sciences, Vladivostok, 17 Palchevskii Str., 690041, Russia. E-mail address: [email protected] (I.V. Manzhulo).
http://dx.doi.org/10.1016/j.jneuroim.2016.07.015 0165-5728/© 2016 Elsevier B.V. All rights reserved.
Polyunsaturated fatty acids (PUFAs) demonstrating anti-inflammatory and antioxidant activity are of considerable interest (Farooqui et al., 2007; Tabakaeva and Tabakaev, 2015). Among the most abundant omega-3 PUFAs in nervous tissue, docosahexaenoic acid (DHA) is critically important for nervous cell functional integrity (Bazan, 2007; Nakamoto and Nishinaka, 2010). A deficiency of DHA in brain tissue may affect the activities of membrane-bound enzymes, ion channels, and receptors eventually leading to disruption of neurotransmission and disturbances in brain functioning (Horrocks and Farooqui, 2004). Neuroprotectin D1 (NPD1), the bioactive metabolite of DHA, exerts neuroprotective, anti-inflammatory and anti-apoptotic bioactivity (Serhan, 2006; Xu et al., 2013). We hypothesized that DHA analgesic effect may be related to its influence on neuron-astrocyte interactions in SCDH superficial lamina.
2. Material and methods 2.1. Animals Male Wistar rats (240 ± 20 g, age 2 months, n = 28) were used in experiments. The rats were housed 2 to 4 per cage with ad lib access to food and water. Animals were maintained at a constant temperature (23 ± 2 °C) and humidity (55 ± 15%) with a 12-h light/dark cycle (lights on 7:00 a.m.). The rats were handled for 5 min once a day during 5 days before the experiments. All procedures were approved by the Animal Ethics Committee at the A.V. Zhirmunsky Institute of Marine
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Biology, Far Eastern Branch, Russian Academy of Sciences, according to the Laboratory Animal Welfare guide lines. 2.2. Drugs A high-purified docosahexaenoic acid was produced in Laboratory of Pharmacology, A.V. Zhirmunsky Institute of Marine Biology, Far Eastern Branch, Russian Academy of Sciences. Briefly, docosahexaenoic (DHA, 22:6 n3) acid was extracted from squid Berryteuthis magister liver. The process consisted of three steps: (1) concentration of the total polyunsaturated fatty acids (PUFA) with urea; (2) separation of individual FA by iodolactonization method, and (3) obtaining of highly purified DHA by preparative HPLC. The urea/FA ratio during the first stage of manufacturing process was 3:1, the temperature: 0 and − 20 °C, allowing to increase the DHA concentration from 6.5% to 32.0%. The iodolactonization method was used for separation and purification of individual PUFA. The final concentration of DHA was 95.6%. Stock solution of DHA was prepared by dissolving in 0.9% saline (1:2). Animals were subcutaneously injected with DHA emulsion at a dose of 4.5 mg/kg (125 mМ/kg) (volume of injection was 150 μl) daily during 2 weeks after surgery. As a control drug we used nonsteroidal anti-inflammatory drug diclofenac at a dose of 4 mg/kg. The dose was selected according to the manufacturer's instructions (Indus Pharma, India). 2.3. Surgical procedures A chronic sciatic nerve constriction injury as neuropathic pain model was performed as previously described by Bennett and Xie (1988). Briefly, rats were anesthetized with sodium pentobarbital (50 mg/kg, i.p.), and the left common sciatic nerve was exposed at the middle of the thigh. Three loosely constrictive ligatures (3 silk gut sutures) were tied around the nerve with spacing of approximately 2 mm. Animals were divided into 4 groups (n = 28): “control” group – animals injected with saline (n = 7); a “CCI” group – animals with sciatic nerve constriction injury (n = 7); a “CCI + DHA” group (n = 7) – DHA-injected animals with sciatic nerve constriction injury and a “CCI + diclofenac” group (n = 7) – diclofenac-injected animals with sciatic nerve constriction injury. 2.4. Evaluation of weight bearing deficit Hind paw weight bearing difference was studied as described previously by Nakazato-Imasato and Kurebayashi (2009) using incapacitance tester (Columbus Instruments, USA). This test assesses the severity of pain in the injured limb by determining the weight distribution between the hind paws when animal is fixed in testing chamber. Briefly, the rats were placed into the plexiglass chamber with separate sensor panels for the right and left hind paw. The pressure of each paw exerted on the sensor surface was measured within 3 s. Three measurements with the 5 minute intervals were performed for each animal. The weight distribution for the right and left paws (in grams) was expressed as a percentage of total animal hind paw weight distribution. 2.5. Evaluation of cold allodynia Cold/Hot Plate Analgesia Meter (Columbus Instruments, USA) was used for the evaluation of cold allodynia, always accompanying the development of neurogenic pain syndrome (Bennett, 1993). The test was performed in the chamber with acrylic walls (30 cm high) equipped with metal flor plate (30 × 30 cm) cooled to 0°. Intact animals are able to stand on the cooled floor equally distributing the weight between all paws for a long time. When nerve is damaged, the duration of contact with the cold plate is significantly reduced. To quantify this parameter, we recorded the limb retention time while animal was in the test chamber for 1 min. All tests were performed daily after surgery. Each animal was tested three times with 5 min intervals between measurements.
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2.6. Immunohistochemistry Collection of material for subsequent histological investigation was performed on day 28. Rats were anesthetized with an overdose of sodium thiopental and transcardially perfused with 100 ml of ice cold saline (~4 °C) followed by 100 ml cold fixative (4% paraformaldehyde in 0.1 M phosphate buffer (PBS), pH 7.2). Lumbar segment of the spinal cord was removed immediately and postfixed for 6 h at 4 °C in fresh buffered 4% paraformaldehyde. After rinsing the material was transferred to 20% sucrose for 24 h at 4 °C. Thereafter, the samples were transferred to a Neg 50 (Thermo Scientific, USA) medium for a day and transverse 50 μm sections were obtained with a cryostat microtome (НМ525, Thermo Scientific, USA). Free-floating sections were pre-incubated in 3% hydrogen peroxide to block endogenous peroxidase activity prior to immunohistochemical staining. After three washes in PBS, sections were incubated in blocking buffer containing 2% bovine serum albumin solution (Santa Cruz, SC-2323, USA) and 0.25% Triton X-100 (Gerbu, USA) during 60 min. Sections of spinal cord were incubated overnight at 4 °C with primary antibodies: anti-GFAP mouse monoclonal antibodies, 1:200, Vector Laboratories (VP-G805), USA; anti-substance P mouse monoclonal antibodies, 1:2000, Abcam (ab14184), USA; anti-nNOS rabbit polyclonal antibodies, 1:2000, Abcam (ab40662), USA. Appropriate secondary antibodies conjugated to horseradish peroxidase (PI-1000, anti-rabbit; PI-2000 anti-mouse) were used according to the manufacturer's instructions (Vector Laboratories, USA, 1:100). Sections were incubated in a secondary antibody solution for 45 min at room temperature. After washing, sections were treated for 5– 10 min with chromogen (Nova RED substrate kit, Vector Laboratories, SK-4800, USA) to elicit the immunoperoxidase reaction. The slices were washed with PBS, dehydrated and embedded in Dako TolueneFree Mounting Medium (Dako, CS705, USA). Slides were visualized using light microscopy (Axio Scope A1, Carl Zeiss, Germany), and images were captured using a digital camera (AxioCam ICc3, Carl Zeiss, Germany). 2.7. Image analysis Microphotographs of lumbar spinal cord dorsal horn (SCDH) ipsilateral side were captured and stored as TIFF files. The resulting images were processed and analyzed using ImageJ software (NIH, USA). The number of nNOS-positive neurons in superficial lamina of the SCDH was counted in every eighth slice. The number of nNOS immunopositive cells in the lumbar spinal cord dorsal horn lamina I-III per 1 mm3 of slice was calculated. The number of images for counting was at least 70 per group. The area of GFAP-positive astrocytes and SP-immunopositive fibers in lumbar spinal cord dorsal horn lamina I-III immunohistochemical staining was determined using ImageJ software (NIH, USA) (plugin: IHC Toolbox). 2.8. Statistical analysis The data obtained by immunohistochemistry studies and physiological tests were subjected to statistical analysis using one-way ANOVA tests followed by a post hoc Tukey's multiple comparison test. Repeated measures ANOVA was carried out to evaluation of weight bearing deficit. Data were shown as mean ± SEM and p b 0.05 was taken as statistically significant. All statistical tests were performed using the Microsoft Excel software. 3. Results 3.1. Effects of DHA and diclofenac treatment on behavior in CCI The duration of animals' observation in our study was determined by the active manifestation of test parameters; as we wanted demonstrate active action of DHA in the deferred period after drug
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administration. Control and presurgery animals symmetrically distribute their weight on the hind limbs, and weight distribution symmetry changes dynamically during pain development. At 4–7 days after nerve injury, animals in the “CCI” group distribute the weight as follows: 71.6 ± 0.7% (intact limb) and 28.4 ± 0.7% (injured limb). At the same time period group “CCI + DHA” demonstrated weight distribution between intact and injured limb: 66.9 ± 1%:33.1 ± 1%, group “CCI + diclofenac”: 70.1 ± 2%:29.9 ± 2%. Since day 20 more equal weight distribution between two hind limbs was found in all groups. At 26–28 days weight distribution in animals of “CCI” group for intact and injured limbs was 63 ± 1%:37 ± 1%, while in “CCI + DHA” and “CCI + diclofenac” groups these indexes were 64.1 ± 1%:35.9 ± 1% and 60 ± 1%:40 ± 1%, respectively (Fig. 1A). Cold allodynia testing showed the reduction of the limb contact time in the “CCI” group at 4–7 days postsurgery (10.5 ± 1.9 s). This parameter increased sharply at 8–15 days postsurgery (approximately 24 s) and further trended toward a reduction. In group “CCI + DHA” cold allodynia was not observed. In group “CCI + diclofenac” allodynia
symptoms appeared at 4–7 days and increased to 8–11 days, but we did not observe cold allodynia at the end of our experiment (Fig. 1B). Trophic lesions and autotomy of one or more fingers on the denervated limb were identified in 60% of animals in the “CCI” group (Fig. 2). In groups “CCI + DHA” and “CCI + diclofenac” such pathological changes were not observed. 3.2. Effects of DHA on SCDH superficial lamina astrocytes after CCI Astrogliosis is a typical astrocyte response to peripheral nerve injury accompanying by glial fibrillary acidic protein (GFAP) up-regulation (Ren, 2010; Donegan et al., 2013). Reactive astrocytes release nitric oxide, pro-inflammatory and anti-inflammatory cytokines, matrix metalloproteinases, and other factors that can contribute to neuronal ion homeostasis shifts and altering of metabolic and neurotransmitter properties (Hald et al., 2009). Peripheral nerve injury and the treatment by analgesic drugs are followed by astrocytes number and morphology dynamic alterations. Morphology of GFAP-positive cells in spinal cord
Fig. 1. Time course of DHA and diclofenac analgesic effects on CCI-induced behavior in rats. (A) The dynamics of hind limb weight distribution. (B) The dynamics of cold allodynia (the duration of hind limb lifting above the cold plate for the 1 min observation). Dotted line is “control” group. *Significant differences was observed between groups “CCI” and “CCI + DHA”, “CCI + diclofenac” was at similar observation points (p b 0.05).
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surface plates after sciatic nerve injury is characterized by processes retraction and cell bodies hypertrophy (Fig. 3B). These morphological rearrangements were not typical for animals treated with DHA. In control animals reactive astroglia covers 38.8 ± 3.7% of plate surface area. On day 28 after the sciatic nerve ligation the density of GFAPpositive astrocytes in spinal cord surface plates in groups “CCI” and “CCI + diclofenac” increased to 49.9 ± 1.5% and 49.8 ± 2%, respectively. The difference between “CCI + DHA” and control groups was not significant (Fig. 3 C, D, E). 3.3. Increasing of SCDH superficial lamina SP-immuno-positive staining in CCI-animals after DHA treatment
Fig. 2. Denervated limb of animal in the “CCI” group. White arrow - trophic ulcer, black arrow - autotomy finger.
Substance P is one of the most important mediators of neuropathic inflammatory pain, taking part in the synthesis and release of numerous pro-nociceptive and pro-inflammatory mediators. In the central nervous system SP plays an important role in the spinal cord neurons sensitization, while in the peripheral nervous system, it is involved in the
Fig. 3. Distribution of GFAP-positive astroglia in the spinal cord dorsal horn in animals with neuropathic pain. (A) “control” group; (B) “CCI” group; (C) “CCI + DHA” group; (D) “CCI + diclofenac” group; (E) astroglia activity dynamics. *Significant differences were observed between group “control” and “CCI”, “CCI + diclofenac” at similar observation points (p b 0.05). + Significant differences between groups “CCI” and “CCI + DHA” were at similar observation points (p b 0.05). #Significant differences between groups “CCI + DHA” and “CCI + diclofenac” were at similar observation points (p b 0.05). Scale bar is 200 μm.
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vasodilatation, decreasing of nociceptive activity threshold, as well as in the maintenance of neurogenic inflammation (Maggi, 1995). Thus, modulation of substance P production in the central and peripheral nervous system is suggested to be determinative way of neuropathic pain management and suppression. Accumulations of tightly interwoven SP-positive fibers were observed in the spinal cord surface plates of animals from all experimental groups (Fig. 4). The area of SP-positive fibers in the spinal cord surface plates was 35.9 ± 2% in control animals. SP-immunoreactivity of spinal the cord surface plates in “CCI” and “CCI + diclofenac” animals at day 28 after surgery was increased to 43.36 ± 1% and 42.7 ± 1.9%, respectively (Fig. 4 A, B, D). The area of immunopositive SP-fibers in the group “CCI + DHA” remained at the level of the «control» (Fig. 4 C, E).
Cizkova, 2000) and antinociception (Duarte et al., 1992). Nitric oxide synthesis system is highly dynamic and constantly undergoes quantitative and qualitative changes in pain conditions (Leonard et al., 2001). NO release during the activation of neuronal and inducible NO-synthase underlies the behavioral manifestations of pain such as cold allodynia and thermal hyperalgesia (Onal et al., 2003). In the control group the number of nNOS-positive neurons in the spinal cord surface plates was 2154.5 ± 221.3 per 1 mm3 (Fig. 5 A). Sciatic nerve damage in the “CCI” group was accompanied by the number of neurons expressing NO-synthase increase to 3245.4 ± 166.6 (Fig. 5 B). The use of DHA and diclofenac prevented the increase of nNOS-positive neurons number. Thus, the number of neurons in groups “CCI + DHA” and “CCI + diclofenac” was 1749.3 ± 235.7 and 1962.2 ± 174.4 1 mm3, respectively (Fig. 5 C, D, E).
3.4. nNOS-positive neurons in SCDH superficial lamina in CCI-animals after DHA treatment
4. Discussion
Pharmacological correction of nitric oxide (NO) release significantly influence the nervous system functioning, including neuromodulation (Kiss, 2000), synaptic plasticity (Holscher, 1997), pain (Luo and
Our data demonstrate that CCI in rats is accompanied by cold allodynia and weight distribution asymmetry as well as degenerative changes in denervated limb tissues. These pathologic changes occur at
Fig. 4. Distribution of SP-positive presynaptic sensory fibers in the spinal cord dorsal horn of animals with neuropathic pain. (A) “control” group; (B) “CCI” group; (C) “CCI + DHA” group; (D) “CCI + diclofenac” group; (E) SP activity dynamics. ⁎Significant differences between groups “control” and “CCI” were observed, “CCI + diclofenac” were at similar observation points (p b 0.05). + Significant differences between groups “CCI” and “CCI + DHA” were at similar observation points (p b 0.05). Scale bar is 200 μm.
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Fig. 5. nNOS-positive neurons in the spinal cord dorsal horns of animals with neuropathic pain. (A) “control” group; (B) “CCI” group; (C) “CCI + DHA” group; (D) “CCI + diclofenac” group; (E) nNOS-positive neuron activity dynamics. ⁎Significant differences between groups “control” and “CCI” were at similar observation points (p b 0.05). + Significant differences between groups “CCI” and “CCI + DHA” were observed, “CCI + diclofenac” were at similar observation points (p b 0.05). Scale bar is 200 μm.
day 4 and have been observed for 28 days after surgery gradually decreasing. The absence of cold allodynia 4 weeks after CCI is not an indicator of pain suppression, as other manifestations of pathological sensitivity (tactile allodynia and thermal hyperalgesia, phantom pain) are also present and even become more intense (Malmberg and Basbaum, 1998). Here we found that neuropathic pain development causes cellular changes in the SCDH superficial lamina. On day 28 after CCI modeling we observed astroglial reactivation accompanied by an increase in neurokinin system activity and enhancement of NO-ergic neurotransmission. We assume that SP immunoreactivity increase is related to peripheral sensitization development as well as with the uncontrolled axonal growth at the site of damage, neurotrophic factors production and neuromas formation (Zimmermann, 2001). Our results demonstrate that activation of neurokinin neurotransmission is dynamically coordinated with the astrocytic glia activation in the spinal cord posterior horn. However, the detailed characteristics of SP-ergic system and spinal cord astrocytes interactions are have to be determined. Nevertheless, it is known that astrocytes express surface neurokinin receptors and get direct projections from SP-ergic afferents (Yihong et al.,
2006) and can be activated by neurokinin afferent signaling. In turn, the release of SP by axons and the activity of the neurokinin system in pain may be under the influence of astrocytes - pharmacological inhibition of its activity is accompanied by a decrease in pain through inhibiting of SP synthesis and release (Guo et al., 2007). In this study, the behavioral testing of cold allodynia did not reveal any direct relationship between astrocytic and neurokinin activity in the spinal cord posterior horn and the severity of the pain behavior in the experimental animals. A number of studies demonstrated that the pharmacological regulation of astrocytes activity, as well as inhibition of SP-ergic neurotransmission is capable of tactile allodynia prevention (Nichols et al., 1999; Clark et al., 2007). Thus, the increase of astrocytes and neurokinin system activity at day 28 after surgery may be related to the development of tactile allodynia. Increased NO-ergic neurotransmission is repeatedly described on various experimental models (Meller et al., 1992; Costigan et al., 2009; Keilhoff et al., 2013) and our findings are broadly consistent with the existing experimental and clinical observations. In our study, the administration of DHA is resulted in a stabilization of the earlier weight distribution and prevents the development of cold
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allodynia and dystrophic changes in the denervated limb tissues. Most authors explain this effect by systemic anti-inflammatory and antioxidant activity of DHA (Bazan, 2007; Cunnane et al., 2009). However, the weight distribution at the end of the observation period in group “CCI + DHA” did not differ significantly from group “CCI”, possibly due to termination of drug administration at day 14 after surgery. On day 28 after the surgery, we found no significant differences in such indicators as cold allodynia and weight distribution between the “CCI” and “CCI + DHA” groups. However, at day 28 DHA reduces the level of SP- and NO-ergic neurotransmitter systems activity and astrocytosis in SCDH superficial lamina to the control group level. Some mechanisms responsible for DHA analgesic activity could be suggested. Here, along with suppression of astrocytosis, DHA possibly inhibit the release of neurotrophic factors and inflammatory mediators by astrocytes which are responsible for neural tissue excitability enhancement and neuropathic pain development (Paterniti et al., 2014; Marques et al., 2015). Furthermore, recent studies demonstrate that PUFA, particularly docosahexaenoic acid is an endogenous ligand of PPAR-α (Peroxisome Proliferator Activated Receptor), expressed constitutively on neurons and astrocytes. Thus, among the possible mechanisms of DHA activity, the authors suggest the activation of PPAR-α, which in turn regulates lipid metabolism, glucose homeostasis, as well as demonstrate anti-inflammatory and anti-proliferative properties (Paterniti et al., 2014). Previously it was found that substance P acts as a mediator of neurogenic inflammation by stimulating the cytokines release in damaged tissues (Wei et al., 2012). Docosahexaenoic acid, in addition to the detected neurokinin neurotransmission inhibition may reduce pro-inflammatory cytokines and chemokines expression by the formation of active metabolite neuroprotectin D1 (Lukiw et al., 2005; Xu et al., 2013). According to our previous study, important factors leading to SP-ergic afferent currents stabilization in animals treated with DHA, is a reduction of apoptosis and gliosis in spinal ganglia neurons (Manzhulo et al., 2015) as well as the inhibition of astrocytosis in the spinal cord posterior horns. Previously, we found that DHA reduces DRG microglia/macrophage activity in animals with CCI (Manzhulo et al., 2015). Since the severity of microglial activation is determined by NO-ergic neurotransmission (Kuboyama et al., 2011) the established reduction of NO-ergic neurons number may indicate a direct inter-modulating effects of microglial cells and NO-ergic nociceptive neurons. This is also confirmed by the fact that in mice knockout for each of the three nitric oxide (NO) synthase (NOS) genes the pain reduction is closely related to microglial response suppression in spinal pain centers (Kuboyama et al., 2011). Diclofenac treatment in CCI animals leads to the weight distribution improvement, reduction of cold allodynia and dystrophic changes in the denervated limb tissues. However, it was less effective the DHA treatment. Nevertheless, this study revealed a decreased nNOS activity in SCDH superficial lamina neurons during DHA and diclofenac administration. Based on this, we assume that it is the inhibition of NO-ergic neurotransmission leads to improved physiological parameters in animals with CCI.
5. Conclusions Our study clearly demonstrates that the activation of neurokinin neurotransmission and nNOS synthesis are dynamically coordinated with the astroglial activation in the SCDH superficial lamina during neuropathic pain development. However, the detailed mechanisms of interaction between SP-, NO-ergic systems and astrocytes of the spinal cord remain to be elucidated. Systemic administration of docosahexaenoic acid to CCI animals reduced the intensity and duration of neurogenic pain syndrome, results in earlier stabilization of weight distribution, prevents the cold allodynia and dystrophic changings in denervated limb tissue. DHA treatment reduced the level of the SP- and NO-ergic neurotransmission and decreased astrocytosis in the SCDH superficial lamina. It can be concluded that DHA analgesic activity in neuropathic
pain is related to suppression reactive astrocyte and the inhibition of SP- and NO-signaling pathways.
Acknowledgements Histological and immunohistochemical studies carried out with the Russian Science Foundation financial support (agreement No. 14-5000034), obtaining DHA and all manipulations with animals and image analysis of the material was funded by RFBR according to the research project No. 16-34-00023 mol_a.
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