Tatsuro Kohno

Partial Peripheral Nerve Injury Promotes a Selective Loss of GABAergic Inhibition in the Superficial Dorsal Horn of the Spinal Cord

To clarify whether inhibitory transmission in the superficial dorsal horn of the spinal cord is reduced after peripheral nerve injury, we have studied synaptic transmission in lamina II neurons of an isolated adult rat spinal cord slice preparation after complete sciatic nerve transection (SNT), chronic constriction injury (CCI), or spared nerve injury (SNI). Fast excitatory transmission remains intact after all three types of nerve injury. In contrast, primary afferent-evoked IPSCs are substantially reduced in incidence, magnitude, and duration after the two partial nerve injuries, CCI and SNI, but not SNT. Pharmacologically isolated GABAAreceptor-mediated IPSCs are decreased in the two partial nerve injury models compared with naive animals. An analysis of unitary IPSCs suggests that presynaptic GABA release is reduced after CCI and SNI. Partial nerve injury also decreases dorsal horn levels of the GABA synthesizing enzyme glutamic acid decarboxylase (GAD) 65 kDa ipsilateral to the injury and induces neuronal apoptosis, detected by terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling staining in identified neurons. Both of these mechanisms could reduce presynaptic GABA levels and promote a functional loss of GABAergic transmission in the superficial dorsal horn.

Ionotropic and Metabotropic Receptors, Protein Kinase A, Protein Kinase C, and Src Contribute to C-Fiber-Induced ERK Activation and cAMP Response Element-Binding Protein Phosphorylation in Dorsal Horn Neurons, Leading to Central Sensitization

Molecular mechanisms underlying C-fiber stimulation-induced ERK (extracellular signal-regulated kinase) activation in dorsal horn neurons and its contribution to central sensitization have been investigated. In adult rat spinal slice preparations, activation of C-fiber primary afferents by a brief exposure of capsaicin produces an eightfold to 10-fold increase in ERK phosphorylation (pERK) in superficial dorsal horn neurons. The pERK induction is reduced by blockade of NMDA, AMPA/kainate, group I metabotropic glutamate receptor, neurokinin-1, and tyrosine receptor kinase receptors. The ERK activation produced by capsaicin is totally suppressed by inhibition of either protein kinase A (PKA) or PKC. PKA or PKC activators either alone or more effectively together induce pERK in superficial dorsal horn neurons. Inhibition of calcium calmodulin-dependent kinase (CaMK) has no effect, but pERK is reduced by inhibition of the tyrosine kinase Src. The induction of cAMP response element binding protein phosphorylation (pCREB) in spinal cord slices in response to C-fiber stimulation is suppressed by preventing ERK activation with the MAP kinase kinase inhibitor 2-(2-diamino-3-methoxyphenyl-4H-1-benzopyran-4-one (PD98059) and by PKA, PKC, and CaMK inhibitors. Similar signaling contributes to pERK induction after electrical stimulation of dorsal root C-fibers. Intraplantar injection of capsaicin in an intact animal increases expression of pCREB, c-Fos, and prodynorphin in the superficial dorsal horn, changes that are prevented by intrathecal injection of PD98059. Intrathecal PD98059 also attenuates capsaicin-induced secondary mechanical allodynia, a pain behavior reflecting hypersensitivity of dorsal horn neurons (central sensitization). We postulate that activation of ionotropic and metabotropic receptors by C-fiber nociceptor afferents activates ERK via both PKA and PKC, and that this contributes to central sensitization through post-translational and CREB-mediated transcriptional regulation in dorsal horn neurons.

Blocking Caspase Activity Prevents Transsynaptic Neuronal Apoptosis and the Loss of Inhibition in Lamina II of the Dorsal Horn after Peripheral Nerve Injury

We show that transsynaptic apoptosis is induced in the superficial dorsal horn (laminas I-III) of the spinal cord by three distinct partial peripheral nerve lesions: spared nerve injury, chronic constriction, and spinal nerve ligation. Ongoing activity in primary afferents of the injured nerve and glutamatergic transmission cause a caspase-dependent degeneration of dorsal horn neurons that is slow in onset and persists for several weeks. Four weeks after spared nerve injury, the cumulative loss of dorsal horn neurons, determined by stereological analysis, is >20%. GABAergic inhibitory interneurons are among the neurons lost, and a marked decrease in inhibitory postsynaptic currents of lamina II neurons coincides with the induction of apoptosis. Blocking apoptosis with the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD) prevents the loss of GABAergic interneurons and the reduction of inhibitory currents. Partial peripheral nerve injury results in pain-like behavioral changes characterized by hypersensitivity to tactile or cold stimuli. Treatment with zVAD, which has no intrinsic analgesic properties, attenuates this neuropathic pain-like syndrome. Preventing nerve injury-induced apoptosis of dorsal horn neurons by blocking caspase activity maintains inhibitory transmission in lamina II and reduces pain hypersensitivity.

Removal of GABAergic inhibition facilitates polysynaptic A fiber-mediated excitatory transmission to the superficial spinal dorsal horn

Primary afferent A-fiber stimulation normally evokes fast mono- or polysynaptic EPSCs of short duration. However, in the presence of the GABA(A) receptor antagonist bicuculline, repetitive, long lasting, polysynaptic EPSCs can be observed following the initial, fast response. A-fiber-induced ERK activation is also facilitated in the presence of bicuculline. The frequency of miniature EPSCs and the amplitude of the monosynaptic A-fiber-evoked EPSCs are not affected by bicuculline or the GABA(A) receptor agonist muscimol, suggesting that GABA(A) receptors located on somatodendritic sites of excitatory interneurons are critical for this action. Bicuculline-enhanced polysynaptic EPSCs are completely eliminated by NMDA receptor antagonists APV and ketamine, as was the augmented ERK activation. This NMDA receptor-dependent phenomenon may contribute to bicuculline-induced allodynia or hyperalgesia, as well as the hypersensitivity observed in neuropathic pain patients.

Peripheral axonal injury results in reduced μ opioid receptor pre- and post-synaptic action in the spinal cord

&NA; In both the spared nerve injury (SNI) and spinal nerve ligation (SNL) rat peripheral neuropathic pain models the presynaptic inhibitory effect of the μ opioid receptor (MOR) agonist (DAMGO) on primary afferent‐evoked excitatory postsynaptic currents (EPSCs) and miniature EPSCs in superficial dorsal horn neurons is substantially reduced, but only in those spinal cord segments innervated by injured primary afferents. The two nerve injury models also reduce the postsynaptic potassium channel opening action of DAMGO on lamina II spinal cord neurons, but again only in segments receiving injured afferent input. The inhibitory action of DAMGO on ERK (extracellular signal‐regulated kinase) activation in dorsal horn neurons is also reduced in affected segments following nerve injury. MOR expression decreases substantially in injured dorsal root ganglion neurons (DRG), while intact neighboring DRGs are unaffected. Decreased activation of MOR on injured primary afferent central terminals and the second order neurons they innervate may minimize any reduction by opioids of the spontaneous pain mediated by ectopic input from axotomized small diameter afferents. Retention of MOR expression and activity in nearby non‐injured afferents will enable, however, an opioid‐mediated reduction of stimulus‐evoked and spontaneous pain carried by intact nociceptor afferents and we find that intrathecal DAMGO (1000 ng) reduces mechanical hypersensitivity in rats with SNL. Axotomy‐induced changes in MOR may contribute to opioid‐ insensitive components of neuropathic pain while the absence of these changes in intact afferents may contribute to the opioid sensitive components.

Peripheral nerve injury alters excitatory synaptic transmission in lamina II of the rat dorsal horn

Using the blind whole cell patch‐clamp recording technique, we investigated peripheral nerve injury‐induced changes in excitatory synaptic transmission to neurones in lamina II of the dorsal horn. Partial (i.e. chronic constriction injury (CCI) and spared nerve injury (SNI)) and complete (i.e. sciatic nerve transection (SNT)) peripheral nerve injury altered the mean threshold intensity for eliciting A fibre‐mediated EPSCs in lamina II neurones. Following SNT and CCI, EPSC threshold was significantly decreased, but following SNI, EPSC threshold was increased (naive: 32 ± 2 μA, SNT: 22 ± 2 μA, CCI: 23 ± 2 μA, SNI: 49 ± 4 μA; P < 0.01, Students unpaired t test). Despite this disparity between models, dorsal root compound action potential recordings revealed no significant difference in the conduction velocity or activation threshold of Aβ and Aδ fibres in naive, SNT, CCI and SNI rats. In addition to the changes in EPSC threshold, we also observed a shift in the distribution of EPSCs. In spinal cord slices from naive rats, polysynaptic Aβ fibre‐evoked EPSCs were observed in 24 % of lamina II neurones, monosynaptic Aδ fibre EPSCs were observed in 34 % and polysynaptic Aδ fibre EPSCs were observed in 7 %. Following SNT and CCI, the percentage of neurones with polysynaptic Aβ fibre EPSCs increased to ⩾ 65 % of the sampled population, while the percentage of neurones with monosynaptic Aδ fibre EPSCs decreased to < 10 %. The percentage of neurones with polysynaptic Aδ fibre EPSCs was unchanged. In contrast, following SNI, Aβ fibre EPSCs decreased in incidence while the percentage of neurones with polysynaptic Aδ fibre EPSCs increased to 44 %. Similar to the other injury models, however, monosynaptic Aδ fibre EPSCs decreased in frequency following SNI. Thus, excitatory synaptic transmission is subject to divergent plasticity in different peripheral nerve injury models, reflecting the complexity of responses to different forms of deafferentation.

Muscarinic facilitation of GABA release in substantia gelatinosa of the rat spinal dorsal horn

1 Blind patch clamp recordings were made from substantia gelatinosa (SG) neurones in the adult rat spinal cord slice to study the mechanisms of cholinergic modulation of GABAergic inhibition. 2 In the majority of SG neurones tested, carbachol (10 μM) increased the frequency (677 % of control) of spontaneous GABAergic inhibitory postsynaptic currents (IPSCs). A portion of these events appeared to result from the generation of spikes by GABAergic interneurones, since large amplitude IPSCs were eliminated by tetrodotoxin (1 μM). 3 The effect of carbachol on spontaneous IPSCs was mimicked by neostigmine, suggesting that GABAergic interneurones are under tonic regulation by cholinergic systems. 4 The frequency of GABAergic miniature IPSCs in the presence of tetrodotoxin (1 μM) was also increased by carbachol without affecting amplitude distribution, indicating that acetylcholine facilitates quantal release of GABA through presynaptic mechanisms. 5 Neither the M1 receptor agonist McN‐A‐343 (10‐300 μM) nor the M2 receptor agonist, arecaidine (10‐100 μM), mimicked the effects of carbachol. All effects of carbachol and neostigmine were antagonized by atropine (1 μM), while pirenzepine (100 nM), methoctramine (1 μM) and hexahydrosiladifenidol hydrochloride, p‐fluoro‐analog (100 nM) had no effect. 6 Focal stimulation of deep dorsal horn, but not dorsolateral funiculus, evoked a similar increase in IPSC frequency to that evoked by carbachol and neostigmine. The stimulation‐induced facilitation of GABAergic transmission lasted for 2‐3 min post stimulation, and the effect was antagonized by atropine (100 nM). 7 Our observations suggest that GABAergic interneurones possess muscarinic receptors on both axon terminals and somatodendritic sites, that the activation of these receptors increases the excitability of inhibitory interneurones and enhances GABA release in SG and that the GABAergic inhibitory system is further controlled by cholinergic neurones located in the deep dorsal horn. Those effects may be responsible for the antinociceptive action produced by the intrathecal administration of muscarinic agonists and acetylcholinesterase inhibitors.

Nerve conduction blockade in the sciatic nerve prevents but does not reverse the activation of p38 mitogen-activated protein kinase in spinal microglia in the rat spared nerve injury model.

Background: Current evidence indicates that p38 mitogen-activated protein kinase activation in spinal microglia contributes to the development of neuropathic pain. However, how nerve injury activates p38 in spinal microglia is incompletely unknown. Nerve injury–induced ectopic spontaneous activity is essential for the generation of neuropathic pain. The authors examined whether peripheral neural activity is necessary for p38 activation in spinal microglia. Methods: To examine whether spinal microglia activation depends on peripheral activity in the rat spared nerve injury (SNI) model, the authors blocked conduction in the sciatic nerve before or 2 days after SNI. The block was produced by applying bupivacaine-loaded microspheres above the nerve injury site. The p38 activation was examined by p38 phosphorylation using a phosphorylated p38 antibody, and neuropathic pain-related behavior was evaluated before and after intrathecal infusion of a p38 inhibitor. Results: Three days after SNI, there was a marked p38 activation in the medial two thirds of the dorsal horn, where the injured tibial and peroneal nerves terminated and where isolectin B4 staining was lost. Phosphorylated p38 was only colocalized with the microglial surface marker OX-42, indicating a microglial localization of phosphorylated p38 in the SNI model. Bupivacaine microspheres produced persistent block (loss of sensory and motor function) of the sciatic nerve for the whole period of the study (3 days). This blockade prevented but did not reverse p38 activation in spinal microglia. Intrathecal infusion of the p38 inhibitor FR167653 prevented and reversed mechanical allodynia on post-SNI day 3. Conclusions: After nerve injury, activity in the peripheral nerve is required for the induction but not the maintenance of p38 activation in spinal microglia.

Cyclooxygenase 2 expression in the spared nerve injury model of neuropathic pain

Cyclooxygenase-2 (COX-2) after induction peripherally, and within the CNS, plays an important role in producing inflammatory pain. However, its role in neuropathic pain models is controversial. Recently a robust and persistent model of partial nerve injury pain, the spared nerve injury (SNI) model, has been developed. The aim of the present study was to examine the regulation of COX-2 in the rat SNI model and to evaluate the effectiveness of the selective COX-2 inhibitor rofecoxib in preventing neuropathic allodynia and hyperalgesia. RNase protection assays revealed only a very small and transient increase in COX-2 mRNA in the dorsal horn of the spinal cord in the SNI model with a maximum change at 24 h. Immunohistochemical analysis showed a small increase in COX-2 protein in the deep layers of the dorsal horn 10 h following SNI surgery. Rofecoxib (100 microM) did not affect spontaneous excitatory postsynaptic currents or alpha-amino-3-hydroxy-5-methyl-4-isoxazole propanoic acid (AMPA) and N-methyl-d-aspartate (NMDA) responses in lamina II neurons from spinal cords of animals with SNI indicating no detectable action on transmitter release or postsynaptic activity. Furthermore, rofecoxib treatment (1 and 3.2 mg/kg for 5 and 3 days respectively starting on the day of surgery) failed to modify the development of allodynia and hyperalgesia in the SNI model. However, rofecoxib significantly reduced inflammatory hypersensitivity evoked by injection of complete Freunds adjuvant into one hindpaw, indicating that the doses used were pharmacologically active. The pain hypersensitivity produced by the SNI model is not COX-2-dependent.

Bradykinin Produces Pain Hypersensitivity by Potentiating Spinal Cord Glutamatergic Synaptic Transmission

Bradykinin, an inflammatory mediator, sensitizes nociceptor peripheral terminals reducing pain threshold. We now show that the B2 kinin receptor is expressed in rat dorsal horn neurons and that bradykinin, a B2-specific agonist, augments AMPA- and NMDA-induced, and primary afferent-evoked EPSCs, and increases the frequency and amplitude of miniature EPSCs in superficial dorsal horn neurons in vitro. Administration of bradykinin to the spinal cord in vivo produces, moreover, an NMDA-dependent hyperalgesia. We also demonstrate that nociceptive inputs result in the production of bradykinin in the spinal cord and that an intrathecal B2-selective antagonist suppresses behavioral manifestations of central sensitization, an activity-dependent increase in glutamatergic synaptic efficacy. Primary afferent-evoked central sensitization is, in addition, reduced in B2 receptor knock-out mice. We conclude that bradykinin is released in the spinal cord in response to nociceptor inputs and acts as a synaptic neuromodulator, potentiating glutamatergic synaptic transmission to produce pain hypersensitivity.