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Leukotriene enhances NMDA-induced inward currents in dorsal horn neurons of the rat spinal cord after peripheral nerve injury

Leukotriene enhances NMDA-induced inward currents in dorsal horn neurons of the rat spinal cord... Background: LTB4 is classified as a leukotriene (LT ), a group of lipid mediators that are derived from arachidonic acid. It is recognized that leukotrienes are involved in the pathogenesis of many diseases, including peripheral inflamma- tory pain. However, little is known about the effects of leukotrienes on the spinal dorsal horn during neuropathic pain. Previously, we reported that there was increased expression of 5-lipoxygenase (5-LO) at spinal microglia, and the leukotriene B4 receptor 1 (BLT1), a high affinity receptor of LTB4, in spinal neurons in spared nerve injury (SNI) model rats. In the present study, we examined the effects of LTB4 on spinal dorsal horn neurons in both naïve and SNI model rats using patch-clamp methods. Results: Bath application of LTB4 did not change AMPA receptor-mediated spontaneous excitatory postsynaptic currents (sEPSCs) or membrane potentials. However, we found that LTB4 enhanced the amplitude of NMDA receptor- mediated sEPSCs and significantly increased exogenous NMDA-induced inward currents in SNI model rats. This increase of inward currents could be inhibited by a selective LTB4 antagonist, U75302, as well as a GDP-β-S, a G-pro- tein inhibitor. These results indicate that both increased LTB4 from spinal microglia or increased BLT1 in spinal neurons after peripheral nerve injury can enhance the activity of NMDA receptors through intracellular G-proteins in spinal dorsal horn neurons. Conclusion: Our findings showed that LTB4, which may originate from microglia, can activate BLT1 receptors which are expressed on the membrane of spinal dorsal horn neurons during neuropathic pain. This glia-neuron interaction induces the enhancement of NMDA currents through intracellular G-proteins. The enhancement of NMDA receptor sensitivity of dorsal horn neurons may lead to central sensitization, leading to mechanical pain hypersensitivity. Background in nociceptive dorsal horn neurons [4–8]. Recently, Nociceptive pathways are recognized to be dynami- lipid mediators, their receptors, and proinflammatory cally modulated by gene expression, protein synthesis, cytokines have become considered some of most inter- and intracellular signaling after peripheral nerve injury esting molecules in pain research [9–11]. Accumulating [1–3]. Particularly, activated glial cells in the spinal cord evidence suggests that lipid mediators, such as prosta- after peripheral nerve injury produce and release pro- glandins (PG), lysophosphatidic acid, platelet-activating inflammatory cytokines, such as interleukin-1 beta factor, and their receptors, have critical roles in nocicep- (IL-1β), tumor necrosis factor-alpha (TNF-α), and neu- tive pathways and pathological pain [12–14]. rotrophins, resulting in the enhancement of excitability Leukotrienes (LTs) are a group of lipid mediators derived from arachidonic acid (AA). LTs include sev- eral products catalyzed in the 5-lipoxygenase (5-LO) *Correspondence: noguchi@hyo-med.ac.jp pathway that are then released from the cell membrane. Department of Anatomy and Neuroscience, Hyogo College of Medicine, LTs have a variety of biological actions and have been 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501, Japan Full list of author information is available at the end of the article recognized as important factors in numerous disease © 2015 Kiyoyuki et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Kiyoyuki et al. Mol Pain (2015) 11:53 Page 2 of 13 processes, including allergic diseases (e.g. asthma, atopic BLT1, the receptor of LTB4, increased in laminae III‑IV in SNI dermatitis), local or systemic inflammatory diseases (e.g. model rats rheumatoid arthritis, psoriasis), cancer, and cardiovascu- In order to show that BLT1 mRNA expression is lar diseases [15, 16]. AA is converted to leukotriene A4 increased in the laminae after peripheral nerve injury, (LTA4) which is then converted to LTB4, LTC4, LTD4, we performed RT-PCR and in  situ hybridization histo- or LTE4. These products are known as bioactive leukot - chemistry of dorsal horn tissue. We confirmed that SNI rienes. LTC4, LTD4 and LTE4 are collectively termed induced the expression of BLT1 mRNA significantly ‘cysteinyl leukotrienes’ (CysLTs). LTs act by binding to compared to naïve rats (Fig.  1a). We found a majority of specific receptors which are located on the outer plasma neurons showing apparent increase of BLT1 mRNA were membrane of structural and inflammatory cells [15]. So located in laminae III-IV (Fig. 1b, c). Thus, our next step far, four G-protein coupled receptors have been cloned was to examine the effect of LTB4 on laminae III–IV neu - and characterized as LTs receptors [17–20]. It is recog- rons using patch-clamp recording. nized that the LTB4 receptor 1 (BLT1) has a high affinity for LTB4, but that BLT2 has a low affinity for LTB4 and LTB4 had no effect on AMPA receptor‑mediated many other LTs. Studies have shown that lipid media- neurotransmission in rat spinal dorsal horn neurons tors have a key role in the pain mechanisms of peripheral All the recorded neurons that were tested exhibited inflammation, while other research indicates the involve - spontaneous excitatory postsynaptic currents (sEP- ment of spinal lipoxygenase metabolites in hyperalgesic SCs) at a holding potential (VH) of −70  mV, at which responses [16]. For example, prostaglandin E2(PGE2) no inhibitory postsynaptic currents (IPSCs) were directly depolarizes spinal dorsal horn neurons via the observed because the reversal potential for IPSCs was prostaglandin E receptor 2 (EP2)-like receptor, resulting near −70  mV [27]. To examine the effects of LTB4 on in the enhancement of dorsal horn neuronal excitability α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid [9]. Lysophosphatidic acid may be released in the spinal cord after nerve injury and affect the excitability of dor - sal horn neurons, which may be involved in hyperalgesia after peripheral nerve injury [12, 21]. Some studies indi- cate that LTs and their synthesizing enzymes are present in the central nervous system, including the spinal cord, and play important roles in both normal and pathological states [22–24]. Previously, we reported that BLT1 mRNA was expressed by non-neuronal cells in DRG and that CysLT2 mRNA was preferentially expressed by small DRG neu- rons [25, 26]. Furthermore, we showed that the expres- sion of 5-LO in spinal microglia and BLT1 mRNAs in spinal neurons (especially laminae III–IV) was increased during peripheral nerve injury using immunohistochem- istry, reverse transcription-polymerase chain reaction (RT-PCR), in  situ hybridization histochemistry (ISHH), and behavioral experiments [25]. These findings suggest that LTB4 contributes to the central sensitization of the spinal cord after peripheral nerve injury. However, it is unclear how LTB4 acts on excitatory neurotransmission in the spinal dorsal horn. In the present study, we exam- ine how LTB4 modulates excitatory neurotransmission in Fig. 1 BLT1 mRNA increased in dorsal horn neurons after peripheral the spinal dorsal horn after nerve injury. nerve injury. a Expression of BLT1 mRNA in the ipsilateral spinal cord following SNI surgery by conventional RT-PCR. Upper gel panels show PCR products from the dorsal horn of the L4–5 spinal cord taken from Results naïve and 7 days after SNI. Lower histogram shows the quantification Whole-cell patch-clamp recordings were made from a of relative BLT1 mRNA levels (mean ± SEM; *, P < 0.05 compared with total of 181 spinal dorsal horn neurons. Stable record- naïve). b, c Bright-field images of in situ hybridization showing BLT1 ings were obtained from slices more than 12  h after the mRNA expression in lamina III of the spinal dorsal horn in naïve (b) dissection, and recordings were made from single dorsal and 7 days after SNI (c). Solid arrowheads indicate the positive cells. Scale bar 25 µm horn neurons for up to 4 h. Kiyoyuki et al. Mol Pain (2015) 11:53 Page 3 of 13 (AMPA) receptor-mediated excitatory synaptic trans- receptor antagonist, and strychnine (2  μM), a glycine mission, LTB4 (10  μM) was dissolved in Krebs solution receptor antagonist, to distinguish it from IPSCs and and was applied by perfusion for 1  min. When the cell AMPA receptor-mediated sEPSCs. In naïve rats the aver- membrane is fixed at −70  mV, N-methyl-d-aspartate age increases in NMDA receptor-mediated sEPSC fre- 2+ (NMDA) receptors are blocked by Mg . The frequency quency and amplitude mediated by LTB4 (10  μM) were and amplitude of sEPSC were not affected by applica - 104.1  ±  7.2  % and 99.8  ±  13.4  % (n  =  5), respectively tion of LTB4 in dorsal horn neurons (n  =  14) of naïve (Fig.  4b). These values were not significantly different rats (Fig.  2a). The frequency and the amplitude were, from control (P  <  0.05). However, we found that LTB4 respectively, 116.1  ±  7.4  % (n  =  14, P  >  0.05) of control slightly enhanced the amplitude of NMDA receptor- (1.2  ±  0.4  Hz) and 105.2  ±  3.9  % (n  =  14, P  >  0.05) of mediated sEPSCs in SNI model rats (Fig. 4a). The average control (8.5 ± 1.8 pA, Fig. 2b). The frequency and ampli - increases in NMDA receptor-mediated sEPSC frequency tude of sEPSC also were not affected by the application and amplitude mediated by LTB4 were 107.8 ± 7.0 % and of LTB4 in dorsal horn neurons (n = 19) of spared nerve 109.6 ± 2.6 % (n = 5), respectively (Fig. 4c). This increase injury (SNI) model rats (Fig.  2c, d). The frequency and in amplitude was significantly large compared to control the amplitude were, respectively, 112.6 ±  9.7  % (n =  19, (P < 0.05). These results suggest LTB4 enhanced postsyn - P  >  0.05) of control (1.8  ±  0.4  Hz) and 103.5  ±  3.6  % aptic NMDA receptor actions in the neuropathic pain (n = 19, P > 0.05) of control (7.1 ± 0.2 pA, Fig. 2d). These model. Therefore, we examined whether LTB4 affects results indicate that LTB4 does not affect AMPA recep - postsynaptic NMDA currents in dorsal horn neurons. tor-mediated sEPSC of dorsal horn neurons in either Exogenous application of NMDA (30  μM) for 30  s naïve or SNI rats. induced inward currents at a holding potential of Next, we investigated whether LTB4 affects AMPA- −50  mV. After 30  s pretreatment of LTB4 (10  μM) in induced currents in dorsal horn neurons. Exogenous naïve rats, NMDA-induced inward currents did not application of AMPA (10  μM) for 30  s induced inward change (n  =  28, Fig.  5a). The average peak amplitude of currents at a holding potential of −70  mV. After 30  s NMDA-induced currents was 103.6  ±  3.9  % of the con- pretreatment of LTB4 (10  μM) in naïve rats, AMPA- trol (8.8  ±  1  pA, Fig.  5b). In the dorsal horn neurons of induced inward currents did not change (n = 11, Fig. 3a). SNI model rats, we found that NMDA-induced currents The average peak amplitude of AMPA-induced currents increased in the presence of LTB4 in 28 neurons among was 100.1 ± 4.9 % of the control (24.5 ± 3.6 pA, Fig. 3b). 91 neurons with NMDA-induced currents (38.1  %, Next, we examined LTB4 effects in SNI model rats. Fig.  5c). The average-peak amplitude of NMDA-induced AMPA-induced currents of SNI model rat also did not currents was 179.3  ±  5.8  % of control (8.8  ±  0.8  pA, change (n  =  13, Fig.  3c). The average peak amplitude of Fig.  5d). These findings suggest that LTB4 enhanced AMPA-induced currents was 102.9 ± 7.4 % of the control NMDA currents in spinal dorsal horn neurons in SNI (24.5 ± 3.6 pA, Fig. 3d). The application of LTB4 did not model rats, but not in the naïve rats. affect AMPA-induced currents of dorsal horn neurons in either naïve or SNI rats. A selective BLT1 antagonist inhibits NMDA currents induced by LTB4 LTB4 enhanced postsynaptic NMDA currents in spinal BLT1 is a high affinity receptor for LTB4. To assess if dorsal horn neurons of SNI model rats the effects of LTB4 on NMDA-induced currents in dor - Application of LTB4 alone had no effect on AMPA recep - sal horn neurons are mediated by BLT1, we investigated tor-mediated excitatory neurotransmission of dorsal the effects of the presence of a selective BLT1 antagonist horn neurons. However, our previous behavioral tests (U75302). In these experiments, U75302 (20  μM) was suggested that LTB4 contributes to neuropathic pain applied 2 min before treatment with LTB4 (10 μM), and [25]. Next, we investigated whether LTB4 was involved the drug remained present while the agonist was being in NMDA receptors response, because NMDA recep- applied and during subsequent stimulation with NMDA tors are well known to contribute to neuropathic pain. (30  μM). The LTB4-induced enhancement of NMDA First, we examined whether LTB4 affects NMDA recep - currents was significantly inhibited by U75302 (n  =  5, tor-mediated sEPSCs. We recorded NMDA receptor- Fig.  6a). The average peak amplitude of NMDA-induced mediated sEPSCs at a holding potential of +40  mV to currents in the absence and the presence of U75302 2+ completely release Mg blockage of NMDA receptors. respectively were 177 ± 14.4 % and 117.6 ± 3.5 % of con- Under this condition, we performed the experiment trol (10.7 ± 2.8 pA, Fig. 6b). In order to exclude the possi- during the simultaneous application of 6-cyano-7-nit- ble non-specific effect of U75302, we found that U75302 roquinoxaline-2,3-dione (CNQX) (10  μM), an AMPA itself had no significant effect on NMDA-induced cur - receptor antagonist, bicuculline (20  μM), a GABA rents (n  =  9, Fig.  6c). The average-peak amplitude of Kiyoyuki et al. Mol Pain (2015) 11:53 Page 4 of 13 Fig. 2 LTB4 had no effects on AMPA receptor-mediated sEPSC in spinal dorsal horn neurons. The frequency and amplitude of sEPSC were not affected by application of LTB4 in dorsal horn neurons of either naïve (a, b) or SNI model rats (c, d). Bar graphs showing the average frequency and amplitude of sEPSC under the treatment with LTB4 (black bar), relative to those before this treatment (control, open bar) in naïve (b) and SNI model rats (d) (naïve; n = 14. SNI; n = 19, mean ± SEM). n.s., not significant Kiyoyuki et al. Mol Pain (2015) 11:53 Page 5 of 13 Fig. 3 LTB4 did not affect AMPA currents in spinal dorsal horn neurons of both naïve and SNI model rats. AMPA-induced currents were not affected by application of LTB4 in either naïve (a) or SNI model rats (c) dorsal horn neurons. b, d Bar graphs showing the average AMPA-induced currents under the treatment with LTB4 (black bar), relative to those before this treatment (control, open bar) in naïve (b) and SNI model rats (d) (naïve; n = 11. SNI; n = 13). n.s., not significant NMDA-induced currents was 92.5  ±  4.5  % of control again applied 30  min later, it significantly suppressed (14.9 ± 1.2 pA, Fig. 6d). These findings suggest that LTB4 NMDA-induced currents (n  =  5, Fig.  7a). The average exerts its actions through BLT1, and enhances NMDA- peak amplitudes of NMDA-induced currents respec- induced currents. tively were 170.2  ±  13.1  % and 113.5  ±  4.8  % of control (8.2  ±  1.1  pA, Fig.  7b). These findings suggest that the Involvement of G‑proteins in the LTB4 enhancement increased NMDA-induced currents by LTB4 were medi- of NMDA currents ated through the activation of G-proteins. To examine the involvement of G-proteins in the increased NMDA-induced current by LTB4, GDP-β-S Discussion (2  mM), a non-hydrolysable analogue of GDP that com- In the present study, we examined the effects of LTB4 in petitively inhibits G-proteins, was added to the pipette the spinal dorsal horn neurons in laminar III–IV using solution. When LTB4 (10  μM) and NMDA (30  μM) whole-cell patch-clamp recording. The main findings were applied just after establishing the whole-cell con- are: (1) The frequency and amplitude of AMPA recep - figuration with pipettes containing potassium glu - tor-mediated sEPSC and AMPA-induced currents in conate and GDP-β-S, NMDA-induced currents were dorsal horn neurons were not affected by application of clearly observed. However, when LTB4 and NMDA were LTB4 in either naïve or SNI model rats. (2) In contrast, Kiyoyuki et al. Mol Pain (2015) 11:53 Page 6 of 13 Fig. 4 The effects of LTB4 on NMDA receptor-mediated sEPSCs. a NMDA receptor-mediated sEPSC has a reversed waveform with slow decay at a holding potential of +40 mV. Three consecutive traces of EPSCs are shown in an expanded scale in time, before (bottom left) and under the action of LTB4 (bottom right). LTB4 slightly enhanced the amplitude of NMDA receptor-mediated sEPSCs in spinal dorsal horn neurons of SNI model rats. b, c Bar graphs showing the average frequency and amplitude of NMDA receptor-mediated sEPSC under the treatment with LTB4 (black bar), relative to those before this treatment (control, open bar) in naïve (b) and SNI model rats (c) (naïve; n = 5. SNI; n = 5). Statistical significance between data shown by bars is indicated by an asterisk; *, P < 0.05; n.s., not significant Kiyoyuki et al. Mol Pain (2015) 11:53 Page 7 of 13 Fig. 5 Enhanced NMDA currents by LTB4 in spinal dorsal horn neurons of SNI model rats. a LTB4 did not change NMDA-induced inward currents in naïve rats (n = 28). b Bar graph showing the average increase rate of NMDA-induced inward currents in naïve rats (open bar; control (NMDA alone), black bar; LTB4 + NMDA) (n = 28). c NMDA-induced inward currents in the presence of LTB4 were enhanced compared to without LTB4. d Bar graph showing the average increase rate of NMDA-induced inward currents in SNI model rats (open bar; control (NMDA alone), black bar; LTB4 + NMDA) (n = 28). *, P < 0.05; n.s., not significant the perfusion of LTB4 enhanced both the amplitude One of the important findings was that the expression of of NMDA receptor-mediated sEPSC and exogenous BLT1 increased in the spinal cord of SNI model rats at NMDA-induced inward currents in spinal dorsal horn 7 days after the peripheral nerve injury. The signal inten - neurons of SNI model rats. However, this enhancement sity of BLT1 mRNA in the dorsal horn was very low in of LTB4 on NMDA responses was not observed in naïve naïve rats and it was significantly elevated in SNI model rats. (3) The increase of NMDA-induced inward currents rats (Fig.  1). Because a majority of neurons expressing was inhibited by a LTB4 antagonist, U75302, as well as a increased BLT1 mRNA were located in laminae III–IV, GDP-β-S, a G-protein inhibitor. we endeavored to record the neuronal activity in laminae We performed blind whole-cell patch-clamp record- III–IV after peripheral nerve injury. ings from laminae III-IV dorsal horn neurons. Expression LTs are lipid mediators with a proinflammatory profile of the LTs receptor had not been examined in the spinal that have been implicated in the pathogenesis of several cord until we recently showed that the subfamily of LTs types of inflammation [28]. For example, the blood and receptors, BLT1 and CysLT1, are expressed in the gray synovial fluids of patients with rheumatoid arthritis con - matter in the spinal cord [25]. BLT1 was localized only tain higher levels of LTB4 than found in healthy subjects in neurons, while CysLT1 was localized in only microglia. [29]. LTB4 is known as a potent neutrophil chemotactic Kiyoyuki et al. Mol Pain (2015) 11:53 Page 8 of 13 Fig. 6 A selective BLT1 antagonist inhibited LTB4-induced enhancement of NMDA currents. a Enhancement of NMDA-currents by LTB4 was signifi- cantly inhibited in the presence of the selective BLT1 antagonist, U75302 (20 μM). b Bar graph showing the average increase rate of NMDA-induced inward currents by NMDA alone as control (open bar) and under the treatment with LTB4 (black bar) and in the presence LTB4 during application U75302 (gray bar), (n = 5). c U75302 had no effect on NMDA-induced currents (n = 9). d Bar graph showing the average increase rate of NMDA- induced inward currents by NMDA alone as control (open bar) and under the treatment with U75302 (black bar). *, P < 0.05 agent. It is believed that in rheumatoid arthritis, neutro- of the pain threshold in humans after intracutaneous phils infiltrate synovial fluids and produce LTB4 inducing deposition of LTB4 [30]. Also, both a LTB4 antagonist the inflammatory condition. Several studies have demon - and BLT1 knockout mice showed reduced pain behav- strated that LTs are involved in peripheral inflammatory iors in inflammatory pain models [33]. It is well known pain [30–32]. Bisgaard et  al. demonstrated a reduction that nerve growth factor (NGF) is up-regulated in Kiyoyuki et al. Mol Pain (2015) 11:53 Page 9 of 13 Fig. 7 Involvement of G-proteins in the LTB4-induced enhancement of NMDA currents. NMDA-induced currents were recorded with a potassium gluconate pipette solution containing GDP-β-S (2 mM). a NMDA-induced currents were recorded with potassium gluconate pipette solution con- taining GDP-β-S. b Bar graphs showing the average increase rate of NMDA-induced inward currents by alone NMDA as control (open bar) and under the treatment with LTB4 (black bar) and in the presence LTB4 after 30 min (gray bar), (n = 5). *, P < 0.05 inflammatory tissue and sensitizes nociceptors [34], lead - horn neurons after SNI, these findings suggest a pos - ing to thermal hyperalgesia [35]. It has also been reported sible interaction of activated microglia and neurons in that NGF increased LTB4 in the rat paw skin. These the spinal cord. The purpose of this study was to dem - results suggest the participation of LTB4 in NGF-induced onstrate whether LTB4 affects the neuronal activity in local thermal hyperalgesia [36]. Other study groups have dorsal horn neurons by using whole cell patch-clamp reported that intrathecal administration of LTB4 leads recordings. We found that bath application of LTB4 did to thermal hyperalgesia [37]. These previous reports not increase AMPA receptor-mediated sEPSCs or mem- indicate that LTs have important roles in chronic pain at brane potential. In contrast, LTB4 significantly enhanced peripheral tissues, especially inflammatory pain. How - both the amplitude of NMDA receptor-mediated sEP- ever, few reports have suggested that LTs are involved in SCs and exogenous NMDA-induced inward currents in the neuropathic pain via central sensitization in the spi- SNI model rats. LTB4 application did not affect sEPSC nal cord. and NMDA-induced currents in naïve rats. It is probably Our previous study showed that 5-LO and FLAP because the expression of the BLT1 in naïve rats was very mRNAs increased in spinal microglia after SNI surgery low as shown in Fig. 1. [25]. 5-LO is the most important enzyme in the synthe- Ionotropic glutamate receptors in the spinal dor- sis of LTs. FLAP enhances the ability of 5-LO to inter- sal horn have emerged as targets of analgesics during act with its substrate. This finding suggests that LTs are the last decade. It is widely known that activation of synthesized in the activated spinal microglia after SNI. NMDA receptors is very important and essential step Together with the increase of BLT1 in spinal dorsal in both initiating and maintaining activity-dependent Kiyoyuki et al. Mol Pain (2015) 11:53 Page 10 of 13 central sensitization and critically contributes to the increase the excitability of dorsal horn neurons in addi- development of pain hypersensitivity after peripheral tion to lipid mediators including LTB4. Total net effects tissue damage or nerve injury [38, 39]. In this study, the on dorsal horn neurons should reflect the changes in pain enhancements of NMDA-induced currents and sEPSC behaviors. by LTB4 were demonstrated using the patch-clamp Under physiological conditions, sensory modalities are technique, which was consistent with the previous mor- associated with dorsal horn lamination. Consistent with phological data [25]. Moreover, the increase of NMDA this pattern of afferent termination, dorsal horn second current was significantly reduced by the BLT1 selec - order neurons in the superficial laminae mainly receive tive antagonist. Together, this data suggests that LTB4 nociceptive input while neurons in deeper dorsal horn increases after peripheral nerve injury, binds BLT1 in laminae mostly convey non-nociceptive (lamina III) or dorsal horn neurons and leads to the enhancement of converged input (laminae IV–VI) [44, 45]. However, in excitatory neurotransmission via modulation of NMDA pathological conditions such as peripheral nerve injury, receptor. previous studies have indicated that laminae III–IV neu- The findings in this study suggest that LTB4 did not rons with dorsal column nuclei showed dynamic changes affect AMPA receptor at either the pre- or the post-syn - and may have a role in the abnormal processing of input aptic sites in the dorsal horn. The reason why LTB4 has in the spinal cord which leads to mechanical hypersen- excitatory effects on NMDA-mediated neuronal activ - sitivity [46–48]. The enhancement of NMDA-medi - ity and not on AMPA-mediated activity is unknown. To ated responses via BLT1 in SNI rats may be involved clarify this point, the precise downstream signaling after in the sensitization in laminae III–IV and result in the BLT1 activation is necessary [40, 41]. Increased NMDA- increase of neuronal activity, leading to neuropathic pain induced currents by LTB4 were completely blocked by behaviors. the G-protein inhibitor GDP-β-S in the pipette solution, Currently, little is known about the mechanisms that suggesting that BLT1 activation by LTB4 could enhance induce LT synthetases in spinal microglia, or what leads the sensitivity of NMDA receptors through intracellular the BLTl to be expressed in dorsal horn neurons. LTB4 G-proteins. This is a fundamental finding and requires in the spinal cord must be an important mediator in neu- more research on the precise mechanisms of signaling ropathic pain, like other lipid mediators, prostaglandins pathways to NMDA receptor subunits. and lysophosphatidic acid [3, 21]. It is also possible that We previously reported that the continuous intrathecal other types of cells in the spinal cord produce LTB4 or injection of 5-LO inhibitors and LTs receptor antagonists other LTs after peripheral nerve injury, because previous significantly suppressed the development of mechani - papers using cell cultures reported the synthesis of LTB4 cal hypersensitivity after SNI, but late application from in astrocytes, oligodendrocytes and endothelical cells 6 days after injury for 1 week did not reverse the mechan- [49–51]. We believe that lipid mediators such as LTB4 ical hypersensitivity [25]. This finding indicates that LTB4 might be important players in neuron-glia interaction has a mechanistic role in neuropathic pain development. and have a role in pain hypersensitivity via activation of In this study, we performed the patch-clamp experiment NMDA-mediated responses in the dorsal horn neuron on the spinal cord of rats 7–10 days after SNI. This timing after peripheral nerve injury. The investigation of LTB4 was set to match the peak time point of increase of the and BLT1 may not only lead to a deeper understanding of expression of the BLT1 receptor in dorsal horn neurons. intractable neuropathic pain, but also have the potential We should note the ineffectiveness in behavioral experi - to find therapeutic agents in the future. ments in  vivo, despite the fact that the BLT1 antagonist significantly suppressed the NMDA-induced current Conclusion in dorsal horn neurons during this time period. It must Our findings showed that LTB4, which may originate be considered that the concentration of the antagonist from microglia, could activate BLT1 receptors, which are in  vivo is uncertain when it is intrathecally delivered. It expressed on the membrane of spinal dorsal horn neu- is not easy to compare the conditions between intrathe- rons during neuropathic pain. LTB4 enhanced both the cal application into living animals and bath application amplitude of NMDA receptor-mediated sEPSC and exog- onto a slice preparation. Another point which should be enous NMDA-induced inward currents in dorsal horn considered is that multiple molecules or pathways may neurons of SNI model rats. The enhancement of NMDA be activated and contribute to the pain hypersensitivity currents is through intracellular G-proteins; however in this period. For example, spinal astrocytes may be an the detailed mechanisms of the downstream signaling important player that release proinflammatory cytokines and the glia-neuron interaction in the spinal cord need and chemokines to enhance and prolong neuropathic further study in order to be clarified. We believe that pain [42, 43]. These molecules from astrocytes might the LTB4-BLT1 mechanism in the spinal cord may be Kiyoyuki et al. Mol Pain (2015) 11:53 Page 11 of 13 involved in central sensitization after peripheral nerve solution saturated with 95 % O and 5 % CO , and main- 2 2 injury. tained at 36 ± 1 °C. The Krebs solution contained the fol - lowing (in mM): 117 NaCl, 3.6 KCl, 2.5 CaCl , 1.2 MgCl , 2 2 Methods 1.2 NaH PO , 25 NaHCO and 11 glucose, pH 7.4. 2 4 3 All of the experimental procedures involving the use of animals were approved by the Ethics Committee on Ani- Patch‑clamp recordings from spinal dorsal horn neurons mal Experiments, Kansai University of Health Sciences Blind whole-cell patch-clamp recordings were made from and the Hyogo College of Medicine Committee on Ani- spinal dorsal horn neurons (lamina III–IV) with patch- mal Research, and were in accordance with the United pipette electrodes having a resistance of 5–10  MΩ. The Kingdom Animals (Scientific Procedures) Act of 1986 patch-pipette solution used to record EPSCs was com- and associated guidelines. Every effort was made to mini - posed of the following (in mM): 135 potassium gluco- mize animal suffering and reduce the number of animals nate, 5 KCl, 0.5 CaCl , 2 MgCl , 5 EGTA, 5 HEPES and 2 2 used. 5 ATP-Mg, pH 7.2. Recording of NMDA receptor-medi- ated sEPSCs was performed using an electrode solution Animal procedures composed of the following (mM): Cs SO 110, tetraeth- 2 4 Male adult Sprague–Dawley rats (4–5 weeks of age, 130– ylammonium 5, CaCl 0.5, MgCl 2, EGTA 5, ATP-Mg 2 2 200  g) were divided into neuropathic pain model and 5 and HEPES–KOH 5; pH 7.2 (305  mOsm). Membrane naïve rat groups. Neuropathic pain model rats were anes- potentials were held at −70  mV in voltage-clamp mode. −1 thetized with sodium pentobarbital (50 mg kg , ip) and After making a gigaohm seal, the membrane patch was received SNI in their hindlimbs [52]. Briefly, the sciatic ruptured by a brief period of more negative pressure, nerve was exposed at the level of its trifurcation into the thus resulting in a whole cell configuration. Signals were sural, tibial, and common peroneal nerves. Each of the acquired with a patch-clamp amplifier (Axopatch 200B; tibial and common peroneal nerves were tightly ligated Molecular Devices, Sunnyvale, CA, USA). Data were by silk and then completely severed in between, leaving digitized with an analog-to-digital converter (Digidata the sural nerve intact. SNI model rats were used in exper- 1440A; Molecular Devices) and stored on a personal iments 7–10 days after surgery. computer using the pCLAMP 10 data acquisition pro- gram (Molecular Devices). In this study, the exogenous Reverse transcription‑polymerase chain reaction (RT‑PCR) NMDA currents were recorded at −50 mV, and the exog- and in situ hybridization histochemistry enous AMPA currents were recorded at −70  mV. The Methods of RT-PCR and in situ hybridization histochem- frequency and amplitude of the sEPSCs when exposed istry were described in detail in our previous paper [25]. to LBT4 were analyzed at −70 mV (AMPA receptor) and +40  mV (NMDA receptor). Data were analyzed using Spinal cord slice preparations Mini Analysis 6.0 software (Synaptosoft, Fort Lee, NJ, The methods used for obtaining adult rat spinal cord USA) and the pCLAMP 10 data acquisition program. slice preparations have been described previously [53]. EPSCs were detected with Mini Analysis by setting the In brief, adult rats were deeply anesthetized with ure- following parameters: amplitude threshold, 5 pA and area thane (1.2  g/kg, IP), and then lumbosacral laminectomy threshold, 20 pA × ms with their fast rise time and decay was performed. The lumbosacral spinal cord (L1–S3) was curve. Spinal dorsal horn neurons were viable for up to removed and placed in pre-oxygenated sucrose-artificial 24  h in slices perfused with pre-oxygenated Krebs solu- cerebrospinal fluid (ACSF) at 1–3 °C. Sucrose-ACSF con - tion, although all of the recordings described here were tained the following (in mM): 223 sucrose, 25 NaHCO , obtained within 12 h. Whole-cell patch-clamp recordings 1.2 NaH PO , 3.6 KCL, 2 CaCl , 1 MgCl , 0.4 ascorbic were stable for up to 4 h. The membrane potentials were 2 4 2 2 acid, 2 pyruvate, 11 glucose and pH 7.4 [54]. Immedi- not corrected for the liquid junction potential between ately after the removal of the spinal cord, the rats were the Krebs and patch-pipette solutions. given an overdose of urethane and were then killed by exsanguination. The pia-arachnoid membrane was Application of the drugs removed after cutting all of the ventral and dorsal roots Drugs were dissolved in Krebs solution and applied near the root entry zone. The spinal cord was mounted by perfusion via a three-way stopcock without any on a microslicer and then a 600-μm-thick transverse change in the perfusion rate or the temperature. The slice was cut from the lumbar segment which mainly L4 time necessary for the solution to flow from the stop - or L5 root entries. The slice was placed on a nylon mesh cock to the surface of the spinal cord was ~30  s. The in the recording chamber, which had a volume of 0.5 ml, drugs used in this study were LTB4 (5S,12R-dihydroxy- and then perfused at a rate of 10–15 ml/min with Krebs 6Z,8E,10E,14Z-eicosatetraenoic acid), BLT1 receptor Kiyoyuki et al. Mol Pain (2015) 11:53 Page 12 of 13 4. DeLeo JA, Yezierski RP. The role of neuroinflammation and neuroimmune antagonist U75302 (6-(6-(3R-hydroxy-1E,5Z-undeca- activation in persistent pain. Pain. 2001;90:1–6. dien-1-yl)-2-pyridinyl)-1,5S-hexanediol) (Sigma, Poole, 5. Milligan ED, Watkins LR. Pathological and protective roles of glia in UK), GDP-β-S (Sigma, St Louis, MO, USA), AMPA, chronic pain. Nat Rev Neurosci. 2009;10:23–36. 6. Scholz J, Woolf CJ. The neuropathic pain triad: neurons, immune cells and NMDA, CNQX, bicuculline, and strychnine (Sigma- glia. Nat Neurosci. 2007;10:1361–8. Aldrich, St. Louis, MO, USA). AMPA and NMDA were 7. Watkins LR, Maier SF. Glia: a novel drug discovery target for clinical pain. first dissolved in distilled water at 1000 times the concen - Nat Rev Drug Discov. 2003;2:973–85. 8. Watkins LR, Milligan ED, Maier SF. Glial activation: a driving force for tration to be used, while CNQX, bicuculline, and strych- pathological pain. Trends Neurosci. 2001;24:450–5. nine were first dissolved in dimethyl sulfoxide at 1000× 9. Ma W, Quirion R. Does COX2-dependent PGE2 play a role in neuropathic the concentration to be used. They were then stored at pain? Neurosci Lett. 2008;437:165–9. 10. Tsuda M, Ishii S, Masuda T, Hasegawa S, Nakamura K, Nagata K, Yamashita −20 °C and were diluted to the desired concentration in T, Furue H, Tozaki-Saitoh H, Yoshimura M, Koizumi S, Shimizu T, Inoue Krebs solution immediately before use. LTB4 and U75302 K. Reduced pain behaviors and extracellular signal-related protein were stored in ethanol at −20 °C. The ethanol was evapo - kinase activation in primary sensory neurons by peripheral tissue injury in mice lacking platelet-activating factor receptor. J Neurochem. rated from the drugs under a gentle stream of nitrogen 2007;102:1658–68. immediately before use. 11. Yao Y, Wolverton JE, Zhang Q, Marathe GK, Al-Hassani M, Konger RL, Trav- ers JB. Ultraviolet B radiation generated platelet-activating factor receptor agonist formation involves EGF-R-mediated reactive oxygen species. J Statistical analysis Immunol. 2009;182:2842–8. All numerical data were expressed as the mean ± S.E.M. 12. 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YK, WT, TN, KN conceived of the project, designed the experiments and 15. Henderson WR Jr. The role of leukotrienes in inflammation. Ann Intern edited the manuscript. KN supervised the project. All authors have joined the Med. 1994;121:684–97. discussion in this project, and contributed to data interpretation. All authors 16. Peters-Golden M, Henderson WR Jr. Leukotrienes. N Engl J Med. read and approved the final manuscript 2007;357:1841–54. 17. Heise CE, O’Dowd BF, Figueroa DJ, Sawyer N, Nguyen T, Im DS, Stocco Author details R, Bellefeuille JN, Abramovitz M, Cheng R, Williams DL Jr, Zeng Z, Liu Q, Department of Anatomy and Neuroscience, Hyogo College of Medicine, Ma L, Clements MK, Coulombe N, Liu Y, Austin CP, George SR, O’Neill GP, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501, Japan. Pain Research Metters KM, Lynch KR, Evans JF. Characterization of the human cysteinyl Center, Kansai University of Health Sciences, 2-11-1 Wakaba Kumatori-cho, leukotriene 2 receptor. 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Latremoliere A, Woolf CJ. Central sensitization: a generator of pain hyper- features of a set of spinal substantia gelatinosa neurons defined by green sensitivity by central neural plasticity. J Pain. 2009;10:895–926. fluorescent protein expression. J Neurosci. 2004;24:836–42. Submit your next manuscript to BioMed Central and take full advantage of:  Convenient online submission  Thorough peer review  No space constraints or color figure charges  Immediate publication on acceptance  Inclusion in PubMed, CAS, Scopus and Google Scholar  Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Molecular Pain Springer Journals

Leukotriene enhances NMDA-induced inward currents in dorsal horn neurons of the rat spinal cord after peripheral nerve injury

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Springer Journals
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Copyright © 2015 by Kiyoyuki et al.
Subject
Medicine & Public Health; Pain Medicine; Molecular Medicine; Neurobiology; Neurosciences; Neurology
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10.1186/s12990-015-0059-5
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Abstract

Background: LTB4 is classified as a leukotriene (LT ), a group of lipid mediators that are derived from arachidonic acid. It is recognized that leukotrienes are involved in the pathogenesis of many diseases, including peripheral inflamma- tory pain. However, little is known about the effects of leukotrienes on the spinal dorsal horn during neuropathic pain. Previously, we reported that there was increased expression of 5-lipoxygenase (5-LO) at spinal microglia, and the leukotriene B4 receptor 1 (BLT1), a high affinity receptor of LTB4, in spinal neurons in spared nerve injury (SNI) model rats. In the present study, we examined the effects of LTB4 on spinal dorsal horn neurons in both naïve and SNI model rats using patch-clamp methods. Results: Bath application of LTB4 did not change AMPA receptor-mediated spontaneous excitatory postsynaptic currents (sEPSCs) or membrane potentials. However, we found that LTB4 enhanced the amplitude of NMDA receptor- mediated sEPSCs and significantly increased exogenous NMDA-induced inward currents in SNI model rats. This increase of inward currents could be inhibited by a selective LTB4 antagonist, U75302, as well as a GDP-β-S, a G-pro- tein inhibitor. These results indicate that both increased LTB4 from spinal microglia or increased BLT1 in spinal neurons after peripheral nerve injury can enhance the activity of NMDA receptors through intracellular G-proteins in spinal dorsal horn neurons. Conclusion: Our findings showed that LTB4, which may originate from microglia, can activate BLT1 receptors which are expressed on the membrane of spinal dorsal horn neurons during neuropathic pain. This glia-neuron interaction induces the enhancement of NMDA currents through intracellular G-proteins. The enhancement of NMDA receptor sensitivity of dorsal horn neurons may lead to central sensitization, leading to mechanical pain hypersensitivity. Background in nociceptive dorsal horn neurons [4–8]. Recently, Nociceptive pathways are recognized to be dynami- lipid mediators, their receptors, and proinflammatory cally modulated by gene expression, protein synthesis, cytokines have become considered some of most inter- and intracellular signaling after peripheral nerve injury esting molecules in pain research [9–11]. Accumulating [1–3]. Particularly, activated glial cells in the spinal cord evidence suggests that lipid mediators, such as prosta- after peripheral nerve injury produce and release pro- glandins (PG), lysophosphatidic acid, platelet-activating inflammatory cytokines, such as interleukin-1 beta factor, and their receptors, have critical roles in nocicep- (IL-1β), tumor necrosis factor-alpha (TNF-α), and neu- tive pathways and pathological pain [12–14]. rotrophins, resulting in the enhancement of excitability Leukotrienes (LTs) are a group of lipid mediators derived from arachidonic acid (AA). LTs include sev- eral products catalyzed in the 5-lipoxygenase (5-LO) *Correspondence: noguchi@hyo-med.ac.jp pathway that are then released from the cell membrane. Department of Anatomy and Neuroscience, Hyogo College of Medicine, LTs have a variety of biological actions and have been 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501, Japan Full list of author information is available at the end of the article recognized as important factors in numerous disease © 2015 Kiyoyuki et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Kiyoyuki et al. Mol Pain (2015) 11:53 Page 2 of 13 processes, including allergic diseases (e.g. asthma, atopic BLT1, the receptor of LTB4, increased in laminae III‑IV in SNI dermatitis), local or systemic inflammatory diseases (e.g. model rats rheumatoid arthritis, psoriasis), cancer, and cardiovascu- In order to show that BLT1 mRNA expression is lar diseases [15, 16]. AA is converted to leukotriene A4 increased in the laminae after peripheral nerve injury, (LTA4) which is then converted to LTB4, LTC4, LTD4, we performed RT-PCR and in  situ hybridization histo- or LTE4. These products are known as bioactive leukot - chemistry of dorsal horn tissue. We confirmed that SNI rienes. LTC4, LTD4 and LTE4 are collectively termed induced the expression of BLT1 mRNA significantly ‘cysteinyl leukotrienes’ (CysLTs). LTs act by binding to compared to naïve rats (Fig.  1a). We found a majority of specific receptors which are located on the outer plasma neurons showing apparent increase of BLT1 mRNA were membrane of structural and inflammatory cells [15]. So located in laminae III-IV (Fig. 1b, c). Thus, our next step far, four G-protein coupled receptors have been cloned was to examine the effect of LTB4 on laminae III–IV neu - and characterized as LTs receptors [17–20]. It is recog- rons using patch-clamp recording. nized that the LTB4 receptor 1 (BLT1) has a high affinity for LTB4, but that BLT2 has a low affinity for LTB4 and LTB4 had no effect on AMPA receptor‑mediated many other LTs. Studies have shown that lipid media- neurotransmission in rat spinal dorsal horn neurons tors have a key role in the pain mechanisms of peripheral All the recorded neurons that were tested exhibited inflammation, while other research indicates the involve - spontaneous excitatory postsynaptic currents (sEP- ment of spinal lipoxygenase metabolites in hyperalgesic SCs) at a holding potential (VH) of −70  mV, at which responses [16]. For example, prostaglandin E2(PGE2) no inhibitory postsynaptic currents (IPSCs) were directly depolarizes spinal dorsal horn neurons via the observed because the reversal potential for IPSCs was prostaglandin E receptor 2 (EP2)-like receptor, resulting near −70  mV [27]. To examine the effects of LTB4 on in the enhancement of dorsal horn neuronal excitability α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid [9]. Lysophosphatidic acid may be released in the spinal cord after nerve injury and affect the excitability of dor - sal horn neurons, which may be involved in hyperalgesia after peripheral nerve injury [12, 21]. Some studies indi- cate that LTs and their synthesizing enzymes are present in the central nervous system, including the spinal cord, and play important roles in both normal and pathological states [22–24]. Previously, we reported that BLT1 mRNA was expressed by non-neuronal cells in DRG and that CysLT2 mRNA was preferentially expressed by small DRG neu- rons [25, 26]. Furthermore, we showed that the expres- sion of 5-LO in spinal microglia and BLT1 mRNAs in spinal neurons (especially laminae III–IV) was increased during peripheral nerve injury using immunohistochem- istry, reverse transcription-polymerase chain reaction (RT-PCR), in  situ hybridization histochemistry (ISHH), and behavioral experiments [25]. These findings suggest that LTB4 contributes to the central sensitization of the spinal cord after peripheral nerve injury. However, it is unclear how LTB4 acts on excitatory neurotransmission in the spinal dorsal horn. In the present study, we exam- ine how LTB4 modulates excitatory neurotransmission in Fig. 1 BLT1 mRNA increased in dorsal horn neurons after peripheral the spinal dorsal horn after nerve injury. nerve injury. a Expression of BLT1 mRNA in the ipsilateral spinal cord following SNI surgery by conventional RT-PCR. Upper gel panels show PCR products from the dorsal horn of the L4–5 spinal cord taken from Results naïve and 7 days after SNI. Lower histogram shows the quantification Whole-cell patch-clamp recordings were made from a of relative BLT1 mRNA levels (mean ± SEM; *, P < 0.05 compared with total of 181 spinal dorsal horn neurons. Stable record- naïve). b, c Bright-field images of in situ hybridization showing BLT1 ings were obtained from slices more than 12  h after the mRNA expression in lamina III of the spinal dorsal horn in naïve (b) dissection, and recordings were made from single dorsal and 7 days after SNI (c). Solid arrowheads indicate the positive cells. Scale bar 25 µm horn neurons for up to 4 h. Kiyoyuki et al. Mol Pain (2015) 11:53 Page 3 of 13 (AMPA) receptor-mediated excitatory synaptic trans- receptor antagonist, and strychnine (2  μM), a glycine mission, LTB4 (10  μM) was dissolved in Krebs solution receptor antagonist, to distinguish it from IPSCs and and was applied by perfusion for 1  min. When the cell AMPA receptor-mediated sEPSCs. In naïve rats the aver- membrane is fixed at −70  mV, N-methyl-d-aspartate age increases in NMDA receptor-mediated sEPSC fre- 2+ (NMDA) receptors are blocked by Mg . The frequency quency and amplitude mediated by LTB4 (10  μM) were and amplitude of sEPSC were not affected by applica - 104.1  ±  7.2  % and 99.8  ±  13.4  % (n  =  5), respectively tion of LTB4 in dorsal horn neurons (n  =  14) of naïve (Fig.  4b). These values were not significantly different rats (Fig.  2a). The frequency and the amplitude were, from control (P  <  0.05). However, we found that LTB4 respectively, 116.1  ±  7.4  % (n  =  14, P  >  0.05) of control slightly enhanced the amplitude of NMDA receptor- (1.2  ±  0.4  Hz) and 105.2  ±  3.9  % (n  =  14, P  >  0.05) of mediated sEPSCs in SNI model rats (Fig. 4a). The average control (8.5 ± 1.8 pA, Fig. 2b). The frequency and ampli - increases in NMDA receptor-mediated sEPSC frequency tude of sEPSC also were not affected by the application and amplitude mediated by LTB4 were 107.8 ± 7.0 % and of LTB4 in dorsal horn neurons (n = 19) of spared nerve 109.6 ± 2.6 % (n = 5), respectively (Fig. 4c). This increase injury (SNI) model rats (Fig.  2c, d). The frequency and in amplitude was significantly large compared to control the amplitude were, respectively, 112.6 ±  9.7  % (n =  19, (P < 0.05). These results suggest LTB4 enhanced postsyn - P  >  0.05) of control (1.8  ±  0.4  Hz) and 103.5  ±  3.6  % aptic NMDA receptor actions in the neuropathic pain (n = 19, P > 0.05) of control (7.1 ± 0.2 pA, Fig. 2d). These model. Therefore, we examined whether LTB4 affects results indicate that LTB4 does not affect AMPA recep - postsynaptic NMDA currents in dorsal horn neurons. tor-mediated sEPSC of dorsal horn neurons in either Exogenous application of NMDA (30  μM) for 30  s naïve or SNI rats. induced inward currents at a holding potential of Next, we investigated whether LTB4 affects AMPA- −50  mV. After 30  s pretreatment of LTB4 (10  μM) in induced currents in dorsal horn neurons. Exogenous naïve rats, NMDA-induced inward currents did not application of AMPA (10  μM) for 30  s induced inward change (n  =  28, Fig.  5a). The average peak amplitude of currents at a holding potential of −70  mV. After 30  s NMDA-induced currents was 103.6  ±  3.9  % of the con- pretreatment of LTB4 (10  μM) in naïve rats, AMPA- trol (8.8  ±  1  pA, Fig.  5b). In the dorsal horn neurons of induced inward currents did not change (n = 11, Fig. 3a). SNI model rats, we found that NMDA-induced currents The average peak amplitude of AMPA-induced currents increased in the presence of LTB4 in 28 neurons among was 100.1 ± 4.9 % of the control (24.5 ± 3.6 pA, Fig. 3b). 91 neurons with NMDA-induced currents (38.1  %, Next, we examined LTB4 effects in SNI model rats. Fig.  5c). The average-peak amplitude of NMDA-induced AMPA-induced currents of SNI model rat also did not currents was 179.3  ±  5.8  % of control (8.8  ±  0.8  pA, change (n  =  13, Fig.  3c). The average peak amplitude of Fig.  5d). These findings suggest that LTB4 enhanced AMPA-induced currents was 102.9 ± 7.4 % of the control NMDA currents in spinal dorsal horn neurons in SNI (24.5 ± 3.6 pA, Fig. 3d). The application of LTB4 did not model rats, but not in the naïve rats. affect AMPA-induced currents of dorsal horn neurons in either naïve or SNI rats. A selective BLT1 antagonist inhibits NMDA currents induced by LTB4 LTB4 enhanced postsynaptic NMDA currents in spinal BLT1 is a high affinity receptor for LTB4. To assess if dorsal horn neurons of SNI model rats the effects of LTB4 on NMDA-induced currents in dor - Application of LTB4 alone had no effect on AMPA recep - sal horn neurons are mediated by BLT1, we investigated tor-mediated excitatory neurotransmission of dorsal the effects of the presence of a selective BLT1 antagonist horn neurons. However, our previous behavioral tests (U75302). In these experiments, U75302 (20  μM) was suggested that LTB4 contributes to neuropathic pain applied 2 min before treatment with LTB4 (10 μM), and [25]. Next, we investigated whether LTB4 was involved the drug remained present while the agonist was being in NMDA receptors response, because NMDA recep- applied and during subsequent stimulation with NMDA tors are well known to contribute to neuropathic pain. (30  μM). The LTB4-induced enhancement of NMDA First, we examined whether LTB4 affects NMDA recep - currents was significantly inhibited by U75302 (n  =  5, tor-mediated sEPSCs. We recorded NMDA receptor- Fig.  6a). The average peak amplitude of NMDA-induced mediated sEPSCs at a holding potential of +40  mV to currents in the absence and the presence of U75302 2+ completely release Mg blockage of NMDA receptors. respectively were 177 ± 14.4 % and 117.6 ± 3.5 % of con- Under this condition, we performed the experiment trol (10.7 ± 2.8 pA, Fig. 6b). In order to exclude the possi- during the simultaneous application of 6-cyano-7-nit- ble non-specific effect of U75302, we found that U75302 roquinoxaline-2,3-dione (CNQX) (10  μM), an AMPA itself had no significant effect on NMDA-induced cur - receptor antagonist, bicuculline (20  μM), a GABA rents (n  =  9, Fig.  6c). The average-peak amplitude of Kiyoyuki et al. Mol Pain (2015) 11:53 Page 4 of 13 Fig. 2 LTB4 had no effects on AMPA receptor-mediated sEPSC in spinal dorsal horn neurons. The frequency and amplitude of sEPSC were not affected by application of LTB4 in dorsal horn neurons of either naïve (a, b) or SNI model rats (c, d). Bar graphs showing the average frequency and amplitude of sEPSC under the treatment with LTB4 (black bar), relative to those before this treatment (control, open bar) in naïve (b) and SNI model rats (d) (naïve; n = 14. SNI; n = 19, mean ± SEM). n.s., not significant Kiyoyuki et al. Mol Pain (2015) 11:53 Page 5 of 13 Fig. 3 LTB4 did not affect AMPA currents in spinal dorsal horn neurons of both naïve and SNI model rats. AMPA-induced currents were not affected by application of LTB4 in either naïve (a) or SNI model rats (c) dorsal horn neurons. b, d Bar graphs showing the average AMPA-induced currents under the treatment with LTB4 (black bar), relative to those before this treatment (control, open bar) in naïve (b) and SNI model rats (d) (naïve; n = 11. SNI; n = 13). n.s., not significant NMDA-induced currents was 92.5  ±  4.5  % of control again applied 30  min later, it significantly suppressed (14.9 ± 1.2 pA, Fig. 6d). These findings suggest that LTB4 NMDA-induced currents (n  =  5, Fig.  7a). The average exerts its actions through BLT1, and enhances NMDA- peak amplitudes of NMDA-induced currents respec- induced currents. tively were 170.2  ±  13.1  % and 113.5  ±  4.8  % of control (8.2  ±  1.1  pA, Fig.  7b). These findings suggest that the Involvement of G‑proteins in the LTB4 enhancement increased NMDA-induced currents by LTB4 were medi- of NMDA currents ated through the activation of G-proteins. To examine the involvement of G-proteins in the increased NMDA-induced current by LTB4, GDP-β-S Discussion (2  mM), a non-hydrolysable analogue of GDP that com- In the present study, we examined the effects of LTB4 in petitively inhibits G-proteins, was added to the pipette the spinal dorsal horn neurons in laminar III–IV using solution. When LTB4 (10  μM) and NMDA (30  μM) whole-cell patch-clamp recording. The main findings were applied just after establishing the whole-cell con- are: (1) The frequency and amplitude of AMPA recep - figuration with pipettes containing potassium glu - tor-mediated sEPSC and AMPA-induced currents in conate and GDP-β-S, NMDA-induced currents were dorsal horn neurons were not affected by application of clearly observed. However, when LTB4 and NMDA were LTB4 in either naïve or SNI model rats. (2) In contrast, Kiyoyuki et al. Mol Pain (2015) 11:53 Page 6 of 13 Fig. 4 The effects of LTB4 on NMDA receptor-mediated sEPSCs. a NMDA receptor-mediated sEPSC has a reversed waveform with slow decay at a holding potential of +40 mV. Three consecutive traces of EPSCs are shown in an expanded scale in time, before (bottom left) and under the action of LTB4 (bottom right). LTB4 slightly enhanced the amplitude of NMDA receptor-mediated sEPSCs in spinal dorsal horn neurons of SNI model rats. b, c Bar graphs showing the average frequency and amplitude of NMDA receptor-mediated sEPSC under the treatment with LTB4 (black bar), relative to those before this treatment (control, open bar) in naïve (b) and SNI model rats (c) (naïve; n = 5. SNI; n = 5). Statistical significance between data shown by bars is indicated by an asterisk; *, P < 0.05; n.s., not significant Kiyoyuki et al. Mol Pain (2015) 11:53 Page 7 of 13 Fig. 5 Enhanced NMDA currents by LTB4 in spinal dorsal horn neurons of SNI model rats. a LTB4 did not change NMDA-induced inward currents in naïve rats (n = 28). b Bar graph showing the average increase rate of NMDA-induced inward currents in naïve rats (open bar; control (NMDA alone), black bar; LTB4 + NMDA) (n = 28). c NMDA-induced inward currents in the presence of LTB4 were enhanced compared to without LTB4. d Bar graph showing the average increase rate of NMDA-induced inward currents in SNI model rats (open bar; control (NMDA alone), black bar; LTB4 + NMDA) (n = 28). *, P < 0.05; n.s., not significant the perfusion of LTB4 enhanced both the amplitude One of the important findings was that the expression of of NMDA receptor-mediated sEPSC and exogenous BLT1 increased in the spinal cord of SNI model rats at NMDA-induced inward currents in spinal dorsal horn 7 days after the peripheral nerve injury. The signal inten - neurons of SNI model rats. However, this enhancement sity of BLT1 mRNA in the dorsal horn was very low in of LTB4 on NMDA responses was not observed in naïve naïve rats and it was significantly elevated in SNI model rats. (3) The increase of NMDA-induced inward currents rats (Fig.  1). Because a majority of neurons expressing was inhibited by a LTB4 antagonist, U75302, as well as a increased BLT1 mRNA were located in laminae III–IV, GDP-β-S, a G-protein inhibitor. we endeavored to record the neuronal activity in laminae We performed blind whole-cell patch-clamp record- III–IV after peripheral nerve injury. ings from laminae III-IV dorsal horn neurons. Expression LTs are lipid mediators with a proinflammatory profile of the LTs receptor had not been examined in the spinal that have been implicated in the pathogenesis of several cord until we recently showed that the subfamily of LTs types of inflammation [28]. For example, the blood and receptors, BLT1 and CysLT1, are expressed in the gray synovial fluids of patients with rheumatoid arthritis con - matter in the spinal cord [25]. BLT1 was localized only tain higher levels of LTB4 than found in healthy subjects in neurons, while CysLT1 was localized in only microglia. [29]. LTB4 is known as a potent neutrophil chemotactic Kiyoyuki et al. Mol Pain (2015) 11:53 Page 8 of 13 Fig. 6 A selective BLT1 antagonist inhibited LTB4-induced enhancement of NMDA currents. a Enhancement of NMDA-currents by LTB4 was signifi- cantly inhibited in the presence of the selective BLT1 antagonist, U75302 (20 μM). b Bar graph showing the average increase rate of NMDA-induced inward currents by NMDA alone as control (open bar) and under the treatment with LTB4 (black bar) and in the presence LTB4 during application U75302 (gray bar), (n = 5). c U75302 had no effect on NMDA-induced currents (n = 9). d Bar graph showing the average increase rate of NMDA- induced inward currents by NMDA alone as control (open bar) and under the treatment with U75302 (black bar). *, P < 0.05 agent. It is believed that in rheumatoid arthritis, neutro- of the pain threshold in humans after intracutaneous phils infiltrate synovial fluids and produce LTB4 inducing deposition of LTB4 [30]. Also, both a LTB4 antagonist the inflammatory condition. Several studies have demon - and BLT1 knockout mice showed reduced pain behav- strated that LTs are involved in peripheral inflammatory iors in inflammatory pain models [33]. It is well known pain [30–32]. Bisgaard et  al. demonstrated a reduction that nerve growth factor (NGF) is up-regulated in Kiyoyuki et al. Mol Pain (2015) 11:53 Page 9 of 13 Fig. 7 Involvement of G-proteins in the LTB4-induced enhancement of NMDA currents. NMDA-induced currents were recorded with a potassium gluconate pipette solution containing GDP-β-S (2 mM). a NMDA-induced currents were recorded with potassium gluconate pipette solution con- taining GDP-β-S. b Bar graphs showing the average increase rate of NMDA-induced inward currents by alone NMDA as control (open bar) and under the treatment with LTB4 (black bar) and in the presence LTB4 after 30 min (gray bar), (n = 5). *, P < 0.05 inflammatory tissue and sensitizes nociceptors [34], lead - horn neurons after SNI, these findings suggest a pos - ing to thermal hyperalgesia [35]. It has also been reported sible interaction of activated microglia and neurons in that NGF increased LTB4 in the rat paw skin. These the spinal cord. The purpose of this study was to dem - results suggest the participation of LTB4 in NGF-induced onstrate whether LTB4 affects the neuronal activity in local thermal hyperalgesia [36]. Other study groups have dorsal horn neurons by using whole cell patch-clamp reported that intrathecal administration of LTB4 leads recordings. We found that bath application of LTB4 did to thermal hyperalgesia [37]. These previous reports not increase AMPA receptor-mediated sEPSCs or mem- indicate that LTs have important roles in chronic pain at brane potential. In contrast, LTB4 significantly enhanced peripheral tissues, especially inflammatory pain. How - both the amplitude of NMDA receptor-mediated sEP- ever, few reports have suggested that LTs are involved in SCs and exogenous NMDA-induced inward currents in the neuropathic pain via central sensitization in the spi- SNI model rats. LTB4 application did not affect sEPSC nal cord. and NMDA-induced currents in naïve rats. It is probably Our previous study showed that 5-LO and FLAP because the expression of the BLT1 in naïve rats was very mRNAs increased in spinal microglia after SNI surgery low as shown in Fig. 1. [25]. 5-LO is the most important enzyme in the synthe- Ionotropic glutamate receptors in the spinal dor- sis of LTs. FLAP enhances the ability of 5-LO to inter- sal horn have emerged as targets of analgesics during act with its substrate. This finding suggests that LTs are the last decade. It is widely known that activation of synthesized in the activated spinal microglia after SNI. NMDA receptors is very important and essential step Together with the increase of BLT1 in spinal dorsal in both initiating and maintaining activity-dependent Kiyoyuki et al. Mol Pain (2015) 11:53 Page 10 of 13 central sensitization and critically contributes to the increase the excitability of dorsal horn neurons in addi- development of pain hypersensitivity after peripheral tion to lipid mediators including LTB4. Total net effects tissue damage or nerve injury [38, 39]. In this study, the on dorsal horn neurons should reflect the changes in pain enhancements of NMDA-induced currents and sEPSC behaviors. by LTB4 were demonstrated using the patch-clamp Under physiological conditions, sensory modalities are technique, which was consistent with the previous mor- associated with dorsal horn lamination. Consistent with phological data [25]. Moreover, the increase of NMDA this pattern of afferent termination, dorsal horn second current was significantly reduced by the BLT1 selec - order neurons in the superficial laminae mainly receive tive antagonist. Together, this data suggests that LTB4 nociceptive input while neurons in deeper dorsal horn increases after peripheral nerve injury, binds BLT1 in laminae mostly convey non-nociceptive (lamina III) or dorsal horn neurons and leads to the enhancement of converged input (laminae IV–VI) [44, 45]. However, in excitatory neurotransmission via modulation of NMDA pathological conditions such as peripheral nerve injury, receptor. previous studies have indicated that laminae III–IV neu- The findings in this study suggest that LTB4 did not rons with dorsal column nuclei showed dynamic changes affect AMPA receptor at either the pre- or the post-syn - and may have a role in the abnormal processing of input aptic sites in the dorsal horn. The reason why LTB4 has in the spinal cord which leads to mechanical hypersen- excitatory effects on NMDA-mediated neuronal activ - sitivity [46–48]. The enhancement of NMDA-medi - ity and not on AMPA-mediated activity is unknown. To ated responses via BLT1 in SNI rats may be involved clarify this point, the precise downstream signaling after in the sensitization in laminae III–IV and result in the BLT1 activation is necessary [40, 41]. Increased NMDA- increase of neuronal activity, leading to neuropathic pain induced currents by LTB4 were completely blocked by behaviors. the G-protein inhibitor GDP-β-S in the pipette solution, Currently, little is known about the mechanisms that suggesting that BLT1 activation by LTB4 could enhance induce LT synthetases in spinal microglia, or what leads the sensitivity of NMDA receptors through intracellular the BLTl to be expressed in dorsal horn neurons. LTB4 G-proteins. This is a fundamental finding and requires in the spinal cord must be an important mediator in neu- more research on the precise mechanisms of signaling ropathic pain, like other lipid mediators, prostaglandins pathways to NMDA receptor subunits. and lysophosphatidic acid [3, 21]. It is also possible that We previously reported that the continuous intrathecal other types of cells in the spinal cord produce LTB4 or injection of 5-LO inhibitors and LTs receptor antagonists other LTs after peripheral nerve injury, because previous significantly suppressed the development of mechani - papers using cell cultures reported the synthesis of LTB4 cal hypersensitivity after SNI, but late application from in astrocytes, oligodendrocytes and endothelical cells 6 days after injury for 1 week did not reverse the mechan- [49–51]. We believe that lipid mediators such as LTB4 ical hypersensitivity [25]. This finding indicates that LTB4 might be important players in neuron-glia interaction has a mechanistic role in neuropathic pain development. and have a role in pain hypersensitivity via activation of In this study, we performed the patch-clamp experiment NMDA-mediated responses in the dorsal horn neuron on the spinal cord of rats 7–10 days after SNI. This timing after peripheral nerve injury. The investigation of LTB4 was set to match the peak time point of increase of the and BLT1 may not only lead to a deeper understanding of expression of the BLT1 receptor in dorsal horn neurons. intractable neuropathic pain, but also have the potential We should note the ineffectiveness in behavioral experi - to find therapeutic agents in the future. ments in  vivo, despite the fact that the BLT1 antagonist significantly suppressed the NMDA-induced current Conclusion in dorsal horn neurons during this time period. It must Our findings showed that LTB4, which may originate be considered that the concentration of the antagonist from microglia, could activate BLT1 receptors, which are in  vivo is uncertain when it is intrathecally delivered. It expressed on the membrane of spinal dorsal horn neu- is not easy to compare the conditions between intrathe- rons during neuropathic pain. LTB4 enhanced both the cal application into living animals and bath application amplitude of NMDA receptor-mediated sEPSC and exog- onto a slice preparation. Another point which should be enous NMDA-induced inward currents in dorsal horn considered is that multiple molecules or pathways may neurons of SNI model rats. The enhancement of NMDA be activated and contribute to the pain hypersensitivity currents is through intracellular G-proteins; however in this period. For example, spinal astrocytes may be an the detailed mechanisms of the downstream signaling important player that release proinflammatory cytokines and the glia-neuron interaction in the spinal cord need and chemokines to enhance and prolong neuropathic further study in order to be clarified. We believe that pain [42, 43]. These molecules from astrocytes might the LTB4-BLT1 mechanism in the spinal cord may be Kiyoyuki et al. Mol Pain (2015) 11:53 Page 11 of 13 involved in central sensitization after peripheral nerve solution saturated with 95 % O and 5 % CO , and main- 2 2 injury. tained at 36 ± 1 °C. The Krebs solution contained the fol - lowing (in mM): 117 NaCl, 3.6 KCl, 2.5 CaCl , 1.2 MgCl , 2 2 Methods 1.2 NaH PO , 25 NaHCO and 11 glucose, pH 7.4. 2 4 3 All of the experimental procedures involving the use of animals were approved by the Ethics Committee on Ani- Patch‑clamp recordings from spinal dorsal horn neurons mal Experiments, Kansai University of Health Sciences Blind whole-cell patch-clamp recordings were made from and the Hyogo College of Medicine Committee on Ani- spinal dorsal horn neurons (lamina III–IV) with patch- mal Research, and were in accordance with the United pipette electrodes having a resistance of 5–10  MΩ. The Kingdom Animals (Scientific Procedures) Act of 1986 patch-pipette solution used to record EPSCs was com- and associated guidelines. Every effort was made to mini - posed of the following (in mM): 135 potassium gluco- mize animal suffering and reduce the number of animals nate, 5 KCl, 0.5 CaCl , 2 MgCl , 5 EGTA, 5 HEPES and 2 2 used. 5 ATP-Mg, pH 7.2. Recording of NMDA receptor-medi- ated sEPSCs was performed using an electrode solution Animal procedures composed of the following (mM): Cs SO 110, tetraeth- 2 4 Male adult Sprague–Dawley rats (4–5 weeks of age, 130– ylammonium 5, CaCl 0.5, MgCl 2, EGTA 5, ATP-Mg 2 2 200  g) were divided into neuropathic pain model and 5 and HEPES–KOH 5; pH 7.2 (305  mOsm). Membrane naïve rat groups. Neuropathic pain model rats were anes- potentials were held at −70  mV in voltage-clamp mode. −1 thetized with sodium pentobarbital (50 mg kg , ip) and After making a gigaohm seal, the membrane patch was received SNI in their hindlimbs [52]. Briefly, the sciatic ruptured by a brief period of more negative pressure, nerve was exposed at the level of its trifurcation into the thus resulting in a whole cell configuration. Signals were sural, tibial, and common peroneal nerves. Each of the acquired with a patch-clamp amplifier (Axopatch 200B; tibial and common peroneal nerves were tightly ligated Molecular Devices, Sunnyvale, CA, USA). Data were by silk and then completely severed in between, leaving digitized with an analog-to-digital converter (Digidata the sural nerve intact. SNI model rats were used in exper- 1440A; Molecular Devices) and stored on a personal iments 7–10 days after surgery. computer using the pCLAMP 10 data acquisition pro- gram (Molecular Devices). In this study, the exogenous Reverse transcription‑polymerase chain reaction (RT‑PCR) NMDA currents were recorded at −50 mV, and the exog- and in situ hybridization histochemistry enous AMPA currents were recorded at −70  mV. The Methods of RT-PCR and in situ hybridization histochem- frequency and amplitude of the sEPSCs when exposed istry were described in detail in our previous paper [25]. to LBT4 were analyzed at −70 mV (AMPA receptor) and +40  mV (NMDA receptor). Data were analyzed using Spinal cord slice preparations Mini Analysis 6.0 software (Synaptosoft, Fort Lee, NJ, The methods used for obtaining adult rat spinal cord USA) and the pCLAMP 10 data acquisition program. slice preparations have been described previously [53]. EPSCs were detected with Mini Analysis by setting the In brief, adult rats were deeply anesthetized with ure- following parameters: amplitude threshold, 5 pA and area thane (1.2  g/kg, IP), and then lumbosacral laminectomy threshold, 20 pA × ms with their fast rise time and decay was performed. The lumbosacral spinal cord (L1–S3) was curve. Spinal dorsal horn neurons were viable for up to removed and placed in pre-oxygenated sucrose-artificial 24  h in slices perfused with pre-oxygenated Krebs solu- cerebrospinal fluid (ACSF) at 1–3 °C. Sucrose-ACSF con - tion, although all of the recordings described here were tained the following (in mM): 223 sucrose, 25 NaHCO , obtained within 12 h. Whole-cell patch-clamp recordings 1.2 NaH PO , 3.6 KCL, 2 CaCl , 1 MgCl , 0.4 ascorbic were stable for up to 4 h. The membrane potentials were 2 4 2 2 acid, 2 pyruvate, 11 glucose and pH 7.4 [54]. Immedi- not corrected for the liquid junction potential between ately after the removal of the spinal cord, the rats were the Krebs and patch-pipette solutions. given an overdose of urethane and were then killed by exsanguination. The pia-arachnoid membrane was Application of the drugs removed after cutting all of the ventral and dorsal roots Drugs were dissolved in Krebs solution and applied near the root entry zone. The spinal cord was mounted by perfusion via a three-way stopcock without any on a microslicer and then a 600-μm-thick transverse change in the perfusion rate or the temperature. The slice was cut from the lumbar segment which mainly L4 time necessary for the solution to flow from the stop - or L5 root entries. The slice was placed on a nylon mesh cock to the surface of the spinal cord was ~30  s. The in the recording chamber, which had a volume of 0.5 ml, drugs used in this study were LTB4 (5S,12R-dihydroxy- and then perfused at a rate of 10–15 ml/min with Krebs 6Z,8E,10E,14Z-eicosatetraenoic acid), BLT1 receptor Kiyoyuki et al. Mol Pain (2015) 11:53 Page 12 of 13 4. DeLeo JA, Yezierski RP. The role of neuroinflammation and neuroimmune antagonist U75302 (6-(6-(3R-hydroxy-1E,5Z-undeca- activation in persistent pain. Pain. 2001;90:1–6. dien-1-yl)-2-pyridinyl)-1,5S-hexanediol) (Sigma, Poole, 5. Milligan ED, Watkins LR. 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Tsuda M, Ishii S, Masuda T, Hasegawa S, Nakamura K, Nagata K, Yamashita −20 °C and were diluted to the desired concentration in T, Furue H, Tozaki-Saitoh H, Yoshimura M, Koizumi S, Shimizu T, Inoue Krebs solution immediately before use. LTB4 and U75302 K. Reduced pain behaviors and extracellular signal-related protein were stored in ethanol at −20 °C. The ethanol was evapo - kinase activation in primary sensory neurons by peripheral tissue injury in mice lacking platelet-activating factor receptor. J Neurochem. rated from the drugs under a gentle stream of nitrogen 2007;102:1658–68. immediately before use. 11. Yao Y, Wolverton JE, Zhang Q, Marathe GK, Al-Hassani M, Konger RL, Trav- ers JB. Ultraviolet B radiation generated platelet-activating factor receptor agonist formation involves EGF-R-mediated reactive oxygen species. J Statistical analysis Immunol. 2009;182:2842–8. All numerical data were expressed as the mean ± S.E.M. 12. 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Journal

Molecular PainSpringer Journals

Published: Sep 9, 2015

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