Cord Dorsal Horn Of Rats Research Articles

SNAP25/syntaxin4/VAMP2/Munc18-1 Complexes in Spinal Dorsal Horn Contributed to Inflammatory Pain

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) have been implicated in the trafficking of postsynaptic glutamate receptors, including N-methyl-d-aspartate (NMDA)-subtype glutamate receptors (NMDARs) that are critical for nociceptive plasticity and behavioral sensitization. However, the components of SNAREs complex involved in spinal nociceptive processing remain largely unknown. Here we found that SNAP25, syntaxin4, VAMP2 and Munc18-1 were localized at postsynaptic sites and formed the complex in the superficial lamina of spinal cord dorsal horn of rats. The complex formation between these SNAREs components were accelerated after intraplantar injection of complete Freund’s adjuvant (CFA), pharmacological removal of GABAergic inhibition or activation of NMDAR in intact rats. The increased SNAP25/syntaxin4/VAMP2/Munc18-1 interaction facilitated the surface delivery and synaptic accumulation of NMDAR during inflammatory pain. Disruption of the molecular interaction between SNAP25 with its SNARE partners by using a blocking peptide derived from the C-terminus of SNAP25 effectively repressed the surface and synaptic accumulation of GluN2B-containing NMDARs in CFA-injected rats. This peptide also alleviated inflammatory mechanical allodynia and thermal hypersensitivity. These data suggested that SNAREs complex assembly in spinal cord dorsal horn was involved in the inflammatory pain hypersensitivity through promoting NMDAR synaptic trafficking.

Electroacupuncture Inhibits the Activity of Astrocytes in Spinal Cord in Rats with Visceral Hypersensitivity by Inhibiting P2Y1 Receptor-Mediated MAPK/ERK Signaling Pathway.

Background Irritable bowel syndrome (IBS) is a chronic functional bowel disease characterized by abdominal pain and changes in bowel habits in the absence of organic disease. Electroacupuncture (EA) has been shown to alleviate visceral hypersensitivity (VH) in IBS rat models by inhibiting the activation of astrocytes in the spinal cord. However, the underlying molecular mechanisms mediated by P2Y1 receptor of this effect of electroacupuncture remain unclear. Aim To explore whether EA inhibits the activity of astrocytes in the spinal cord dorsal horn of rat with visceral hypersensitivity by inhibiting P2Y1 receptor and its downstream mitogen activated protein kinase/extracellular regulated kinase 1 (MAPK/ERK) pathway. Methods Ten-day-old Sprague-Dawley (SD) male rats were given an intracolonic injection of 0.2 ml of 0.5% acetic acid (AA) to establish a visceral hypersensitivity model. EA was performed at Zusanli (ST 36) and Shangjuxu (ST 37) at 100 Hz for 1.05 s and 2 Hz for 2.85 s alternately, pulse width for 0.1 ms, 1 mA, 30 min/d, once a day, for 1 week. Cytokines IL-6, IL-1β, and TNF-α were analyzed by ELISA. The expressions of the P2Y1 receptor and pERK1/2 were analyzed by Western Blot and real-time PCR in the model and EA treated animals to explore the molecular mechanism of EA in inhibiting the activity of spinal cord dorsal horn (L6-S2 segment) astrocytes in rats with IBS visceral hypersensitivity. Results EA significantly reduced the behavioral abdominal withdrawal reflex score (AWRs) of IBS rats with visceral hypersensitivity induced by AA. For comparison, intrathecal injection of astrocytes activity inhibitor fluorocitrate (FCA) also reduced visceral hypersensitivity in IBS rats. EA at Zusanli and Shangjuxu inhibited the mRNA and protein expression of the glial fibrillary acidic protein (GFAP) and in rat spinal cord and reduced the release of inflammatory cytokines IL-6, IL-1, and TNF-α were analyzed by ELISA. The expressions of the P2Y1 receptor and pERK1/2 were analyzed by Western Blot and real-time PCR in the model and EA treated animals to explore the molecular mechanism of EA in inhibiting the activity of spinal cord dorsal horn (L6-S2 segment) astrocytes in rats with IBS visceral hypersensitivity. β, and TNF-μg, 10 μg, 10 Conclusion EA inhibited astrocyte activity in the spinal cord dorsal horn of rat with IBS visceral hypersensitivity by inhibiting the P2Y1 receptor and its downstream, PKC, and MAPK/ERK1/2 pathways.

Effect of ropivacaine on peripheral neuropathy in streptozocin diabetes-induced rats through TRPV1-CGRP pathway.

Objective To determine the effect of ropivacaine on peripheral neuropathy in diabetic rats and its possible mechanism.Methods Forty-eight Sprague–Dawley rats were randomly divided into six groups: nondiabetic control group, nondiabetic group A (0.25% ropivacaine), nondiabetic group B (0.75% ropivacaine), diabetic control group (diabetic peripheral neuropathy (DPN) +artificial cerebrospinal fluid), diabetic group A (DPN+0.25% ropivacaine), and diabetic group B (DPN + 0.75% ropivacaine), with eight rats in each group. Within an hour of the last administration, the sciatic motor nerve conduction velocity (MNCV) of each group was measured, and the morphological changes of rat sciatic nerve were observed by HE, Weil’s staining and electron microscopy. The expression of transient receptor potential vanilloid (TRPV1) in the spinal cord dorsal horn of rats was analyzed by immunohistochemistry, and the expression of Calcitonin gene-related peptide (CGRP) protein in the spinal cord was analyzed by Western blot.Results Compared with the nondiabetic control group, elevated blood glucose, decreased weight and reduced average mechanical withdrawal threshold (MWT), additionally, the sciatic nerves showed significantly slowed conduction velocity (both P<0.001) and damaged pathological structure, the expression of TRPV1 and CGRP were decreased (both P<0.001) in the diabetic groups. Compared with the diabetic control group, down-regulation of TRPV1 and CGRP in spinal cord was significant for the diabetic groups A and B treated with 0.25 and 0.75% ropivacaine, the higher concentration of ropivacaine correlated with a greater change.Conclusion Ropivacaine can significantly block sciatic nerve conduction velocity in DPN rats in a concentration-dependent manner, which may be related to the expression of the TRPV1-CGRP pathway.

Inhibition of protein tyrosine phosphatase 1B in spinal cord dorsal horn of rats attenuated diabetic neuropathic pain

Protein tyrosine phosphatase 1B (PTP1B) has been shown to dephosphorylate and inactivate insulin receptors, which contributes to the pathogenesis of diabetes. Neuropathic pain is one of the severe complications that results from diabetic neuropathy. However, whether PTP1B was involved in the development of diabetic neuropathic pain is largely unknown. The current study illustrated that PTP1B was located in spinal cord dorsal horn neurons of Sprague-Dawley rats. Western blot analysis demonstrated that the diabetic neuropathic pain induced by intraperitoneal injection of streptozotocin was associated with an increased protein expression and a dynamic redistribution of spinal PTP1B into excitatory glutamatergic synapses. We found that PTP1B operated to stimulate Src kinase and enhance the tyrosine phosphorylation of N-methyl-D-aspartate (NMDA) subtype of glutamate receptors. The siRNA-mediated knockdown of PTP1B in streptozotocin-injected rats repressed Src activity, decreased NMDA receptor phosphorylation and alleviated the thermal hyperalgesia and mechanical allodynia. A similar analgesia against diabetic neuropathic pain was also achieved when PTP1B activity was manipulated by a chemical PTP Inhibitor or PTP1B(C215S) mutant. These data revealed a regulated expression of PTP1B in spinal cord dorsal horn of rats after diabetic neuropathy, and demonstrated that inhibition of PTP1B was beneficial for the treatment of pain hypersensitivity related to diabetes.

Adenosine A1 receptor potentiated glycinergic transmission in spinal cord dorsal horn of rats after peripheral inflammation

Adenosine is present at the extracellular space within spinal cord dorsal horn and engaged in the processing of nociceptive sensory signals. Systemic or spinal administration of exogenous adenosine produces a potent analgesia against pathological pain. Here we found that inhibitory glycinergic neurotransmission was an important target for adenosine regulation. In spinal cord slices from intact rats, adenosine increased the inhibitory postsynaptic currents mediated by glycine receptors (GlyRs). In spinal slices from Complete Freund's Adjuvant-injected rats, adenosine potentiated glycinergic transmission to a more degree than in control rats. This synaptic potentiation was dependent on the activation of adenosine A1 receptor (A1R), and attributed to the modification of postsynaptic GlyRs function. The Gi protein-coupled A1R typically signals through Gαi/cAMP-dependent protein kinase (PKA) and Gβγ pathways. We found that blockade of either Gαi/PKA or Gβγ signaling attenuated the ability of adenosine to increase glycinergic synaptic responses in inflamed rats. To identify which GlyRs subunit was subjected to A1R regulation, we recorded glycine-evoked whole-cell currents in HEK293T cells co-transfected with A1R and distinct GlyRs subunit. We found that α1, the most abundant functional GlyRs subunit in adult spinal cord, was insensitive to A1R activation. However, when GlyRs α3 subunit or α1ins subunit, a longer α1 isoform, was co-expressed with A1R, adenosine caused a significant increase of glycinergic currents. Inhibition of PKA and Gβγ abolished the stimulatory effects of A1R on α3 and α1ins, respectively. These data suggested that A1R might potentiate glycinergic transmission through Gαi/PKA/α3 and Gβγ/α1ins pathways in inflamed rat.

Striatal-enriched phosphatase 61 inhibited the nociceptive plasticity in spinal cord dorsal horn of rats

Striatal-enriched phosphatase 61 (STEP61) is a member of intracellular protein tyrosine phosphatases, which is involved in the regulation of synaptic plasticity and a line of neuropsychiatric disorders. This protein tyrosine phosphatase is also abundant in pain-related spinal cord dorsal horn neurons. However, whether and how this tyrosine phosphatase modulates the nociceptive plasticity and behavioral hypersensitivity remain largely unknown. The present study recorded the long-term potentiation (LTP) of primary afferent C fiber-evoked field potentials in vivo in superficial dorsal horn of rats, and tested the possible role of STEP61 in spinal LTP. We found that LTP induction significantly increased STEP61 phosphorylation at Ser221 residue, a key molecular event that has been shown to impair the phosphatase activity. The STEP61 hypoactivity allowed for the activation of three substrates, GluN2B subunit-containing N-methyl-d-aspartate-subtype glutamate receptors, Src-family protein tyrosine kinase member Fyn and extracellular signal-regulated kinase 1/2, through which the thresholds for LTP induction were noticeably decreased. To reinstate STEP61 activity, we overexpressed wild-type STEP61 [STEP61(WT)] in spinal dorsal horn, finding that STEP61(WT) completely blunted LTP induction. Behavioral tests showed that LTP blockade by STEP61(WT) correlated with a long-lasting alleviation of thermal hypersensitivity and mechanical allodynia induced by chronic constriction injury of sciatic nerves. These data implicated that STEP61 exerted a negative control over spinal nociceptive plasticity, which might have therapeutic benefit in pathological pain.

Spinophilin-Targeted Protein Phosphatase-1 Alleviated Inflammatory Pain by Negative Control of MEK/ERK Signaling in Spinal Cord Dorsal Horn of Rats.

Extracellular signal-regulated kinase (ERK) relays the signals from multiple transmembrane receptors to a wide range of downstream effectors critical for the regulation of neuronal excitability and plasticity. The strength and duration of ERK signaling is spatiotemporally controlled by protein phosphatases. Sustained activation of ERK has been implicated in a variety of pathological processes. The current study revealed that spinophilin, a well characterized protein phosphatase 1 (PP1) synaptic targeting protein, was able to scaffold mitogen-activated protein kinase kinase (MEK) and ERK for dephosphorylation and inactivation by PP1. The loss of PP1 inhibition, as a result of spinophilin/PP1 dissociation, led to aberrant activation of MEK/ERK signaling, which had important implications for the exaggeration of NMDA receptor-dependent nociceptive synaptic transmission in spinal cord dorsal horn.

A novel therapeutic target for peripheral nerve injury-related diseases: aminoacyl-tRNA synthetases

Aminoacyl-tRNA synthetases (AminoARSs) are essential enzymes that perform the first step of protein synthesis. Beyond their original roles, AminoARSs possess non-canonical functions, such as cell cycle regulation and signal transduction. Therefore, AminoARSs represent a powerful pharmaceutical target if their non-canonical functions can be controlled. Using AminoARSs-specific primers, we screened mRNA expression in the spinal cord dorsal horn of rats with peripheral nerve injury created by sciatic nerve axotomy. Of 20 AminoARSs, we found that phenylalanyl-tRNA synthetase beta chain (FARSB), isoleucyl-tRNA synthetase (IARS) and methionyl-tRNA synthetase (MARS) mRNA expression was increased in spinal dorsal horn neurons on the injured side, but not in glial cells. These findings suggest the possibility that FARSB, IARS and MARS, as a neurotransmitter, may transfer abnormal sensory signals after peripheral nerve damage and become a new target for drug treatment.

Intrathecal Ultra-Low Dose Naloxone Enhances the Antihyperalgesic Effects of Morphine and Attenuates Tumor Necrosis Factor-α and Tumor Necrosis Factor-α Receptor 1 Expression in the Dorsal Horn of Rats with Partial Sciatic Nerve Transection

Glutamate homeostasis and microglia activation play an important role in the development and maintenance of neuropathic pain. We designed this investigation to examine whether ultra-low dose naloxone administered alone or in combination with morphine could alter the concentration of the excitatory amino acids (EAAs) glutamate and aspartate, as well as the expression of tumor necrosis factor-α (TNF-α) and its receptors (TNFR1 and TNFR2) in the spinal cord dorsal horn of rats with partial sciatic nerve transection (PST). Male Wistar rats underwent intrathecal catheter implantation for drug delivery and were divided in 7 groups: sham-operated + saline (sham), PST + saline (S), PST + 15 ng naloxone (n), PST + 15 µg naloxone (N), PST + 10 µg morphine (M), PST + 15 ng naloxone + 10 µg morphine (Mn), PST + 15 µg naloxone + 10 µg morphine (MN). Thermal withdrawal latency and mechanical withdrawal threshold, TNF-α and TNFR expression in the spinal cord and dorsal root ganglia, and EAAs glutamate and aspartate concentration in cerebrospinal fluid dialysates were measured. Ten days after PST, rats developed hyperalgesia (P < 0.0001) and allodynia (P < 0.0001), and increased TNF-α (P < 0.0001) and TNFR1 expression (P = 0.0009) were measured in the ipsilateral spinal cord dorsal horn. The antihyperalgesic and antiallodynic effects of morphine (10 μg) were abolished by high-dose naloxone (15 μg; P = 0.0031) but enhanced by ultra-low dose naloxone (15 ng; P = 0.0015), and this was associated with a reduction of TNF-α (P < 0.0001) and TNFR1 (P = 0.0009) expression in the spinal cord dorsal horn and EAAs concentration (glutamate: P = 0.0001; aspartate: P = 0.004) in cerebrospinal fluid dialysate. Analysis of variance (ANOVA) or Student t test with Bonferroni correction were used for statistical analysis. Ultra-low dose naloxone enhances the antihyperalgesia and antiallodynia effects of morphine in PST rats, possibly by reducing TNF-α and TNFR1 expression, and EAAs concentrations in the spinal dorsal horn. Ultra-low dose naloxone may be a useful adjuvant for increasing the analgesic effect of morphine in neuropathic pain conditions.

Metabotropic glutamate receptors (mGluRs) regulate noxious stimulus‐induced glutamate release in the spinal cord dorsal horn of rats with neuropathic and inflammatory pain

In rats with persistent pain, spinal group I metabotropic glutamate receptor (mGluR) activity has been shown to be pronociceptive, whereas spinal group II/III activity is anti-nociceptive. In brain, group I mGluR activity produces positive feedback effects on glutamate release, whereas group II/III activity produces negative feedback effects. It is unknown whether the nociceptive versus anti-nociceptive effects of spinal group I versus group II/III mGluR activity depend on differential regulation of spinal glutamate release. Here, we used behavioral nociceptive testing and in vivo microdialysis to assess the effect of intrathecal treatment with group I mGluR antagonists [cyclopropan[b] chromen-1a-carboxylate, (CPCCOEt), 2-methyl-6-(phenylethynyl) pyridine (MPEP)] or groups II [aminopyrrolidine-2R,4R-dicarboxylate (APDC)] and III [l-2-amino-4-phosphonobutyrate (l-AP4)] mGluR agonists or vehicle, on nociception and noxious stimulus-induced increases in glutamate release in the spinal cord dorsal horn of rats with a chronic constriction injury (CCI) of the sciatic nerve or hind paw injection of complete Freund's adjuvant (CFA). None of the treatments significantly influenced basal spinal glutamate concentrations in either CCI or CFA rats. In CCI rats, formalin-induced nociception and increases in spinal glutamate concentrations were significantly attenuated by pre-treatment with CPCCOEt, MPEP, APDC, or l-AP4. In CFA rats, capsaicin-induced increases in nociception and spinal glutamate concentrations were significantly attenuated by pre-treatment with CPCCOEt, MPEP, or APDC, but not l-AP4. This study demonstrates that group I antagonists and group II/III mGluR agonists attenuated the enhanced nociception and noxious stimulus-induced glutamate release in spinal cord dorsal horn of CCI and/or CFA rats in vivo, and suggests a possible mechanism for their anti-hyperalgesic effects.

Increases in protein kinase C gamma immunoreactivity in the spinal cord dorsal horn of rats with painful mononeuropathy

Eight days after chronic constrictive sciatic nerve injury (CCI), protein kinase C gamma (PKCγ) immunoreactivity reliably increased in the spinal cord dorsal horn of CCI rats with demonstrable thermal hyperalgesia as compared to sham-operated controls. Such PKCγ immunostaining was observed primarily in neuronal somata (ipsilateral > contralateral, laminae I–II > III–IV), indicating postsynaptic sites of PKCγ increases. Both the development of thermal hyperalgesia and the increase in PKCγ immunoreactivity in CCI rats were prevented by once daily intrathecal administration with 10 nmol MK-801 for 7 days. The present results provide further evidence for a role of PKC in N-methyl- d-aspartate (NMDA) receptor-mediated mechanisms of thermal hyperalgesia.

Increases in protein kinase C gamma immunoreactivity in the spinal cord of rats associated with tolerance to the analgesic effects of morphine

Our previous studies have indicated a critical role of protein kinase C (PKC) in intracellular mechanisms of tolerance to morphine analgesia. In the present experiments, we examined (1) the cellular distribution of a PKC isoform (PKCγ) in the spinal cord dorsal horn of rats associated with morphine tolerance by utilizing an immunocytochemical method and (2) the effects of the N-methyl- d-aspartate receptor antagonist MK-801 on tolerance-associated PKCγ changes. In association with the development of tolerance to morphine analgesia induced by once daily intrathecal administration of 10 μg morphine for eight days, PKCγ immunoreactivity was clearly increased in the spinal cord dorsal horn of these same rats. Within the spinal cord dorsal horn of morphine tolerant rats, there were significantly more PKCγ immunostained neurons in laminae I–II than in laminae III–IV and V–VI. Such PKCγ immunostaining was observed primarily in neuronal somata indicating a postsynaptic site of PKCγ increases. Moreover, both the development of morphine tolerance and the increase in PKCγ immunoreactivity were prevented by co-administration of morphine with 10 nmol MK-801 between Day 2 and Day 7 of the eight day treatment schedule. In contrast, PKCγ immunoreactivity was not increased in rats receiving a single i.t. administration of 10 μg morphine on Day 8, nor did repeated treatment with 10 nmol MK-801 alone change baseline levels of PKCγ immunoreactivity. These results provide further evidence for the involvement of PKC in NMDA receptor-mediated mechanisms of morphine tolerance.

Changes in lectin, GAP-43 and neuropeptide staining in the rat superficial dorsal horn following experimental peripheral neuropathy

The density and distribution of reactivity for two lectins (soybean agglutinin (SBA) and RL-29), growth associated protein-43 (GAP-43) and the neuropeptides substance P and calcitonin gene-related peptide were analyzed in the spinal cord dorsal horn of rats with an experimental peripheral neuropathy. Twenty-eight days postsurgery, the density of label for RL-29 and GAP-43 was increased in laminae I and II on the experimental compared to the control side. In contrast, the density of neuropeptide label was decreased in the same region. Furthermore, on the experimental side, the distribution of both SBA and RL-29 reactivity was increased, extending into lamina III. We hypothesize that the increases in density and distribution of reactivity for the lectins and GAP-43, as well as the decreases in neuropeptide reactivity, reflect injury-induced regenerative changes in primary afferent terminals.

Parvalbumin- and calbindin D28k-immunoreactive neurons in the superficial layers of the spinal cord dorsal horn of rat

Immunohis tochemical techniques were utilized to investigate the distribution and morphology of neurons containing the calcium binding proteins parvalbumin (PV) and calbindin D28k (CaBP) in the superficial layers of rat spinal cord. Most PV-immunoreactive (PV-IR) neurons were restricted to a 25 to 60 μm thick band straddling the border between lamina II and III. Positive somata had long rostrocaudally oriented dendrites confined to narrow sagittally arranged sheets within this band and axons that entered lamina II or the superficial portions of lamina III. Long varicose axons, presumed to originate from these cells, were moderately distributed in Lissauer's tract and lamina II. CaBP-immunoreactive (CaBP-IR) neurons were found within lamina I and throughout lamina II. Large calibre PV-IR and CaBP-IR axons were seen in the dorsal column and the lateral funiculus. Dorsal rhizotomy or neonatal capsaicin treatment appeared to have no effect on PV-IR and CaBP-IR elements in the superficial lumbar dorsal horn. However, dorsal rhizotomy reduced the number of positive axons in the dorsal column and in deeper lamina of the dorsal horn. These results add to the known lamination patterns of the superficial dorsal horn and point to the existence of a lamina defined by PV-positive neurons at the lamina II/III border. These neurons may have electrophysiological characteristics attributed to PV- or CaBP-containing neurons elsewhere in the CNS.

Changes in microglial cell numbers in the spinal cord dorsal horn following brachial plexus transection in the adult rat.

The effect of peripheral nerve transection on the size of the microglial cell population in cytoarchitecturally distinct regions of the spinal cord dorsal horn of rats was evaluated at selected intervals 2 through 35 days after unilateral brachial plexotomy. The identification of cells was verified by electron microscopic examination of a representative random sample of cells included in the counts. Microglial cell numbers were increased in laminae I, II as well as the arbitrarily defined deeper laminae 3.5 days after surgery. Although microglial cell numbers in laminae I were within normal range 35 days after axotomy, those of the more ventrally located laminae remained significantly greater than control values for the duration of the experimental period. These findings demonstrate that: 1) microglial cell proliferation in the dorsal horn is an early event in the central changes that are attendant to peripheral nerve injury 2) the time course of the response varies in cytoarchitecturally different regions.