Ascending pathways for mechanical allodynia in a rat model of neuropathic pain

To determine what the routes by which mechanical allodynia is transmitted following peripheral nerve injury, we assessed the effects of the dorsal column (DC) lesion performed before and 2 weeks after the partial injury of nerves innervating the tail on mechanical allodynia. Ipsilateral DC lesion 2 weeks after neuropathic surgery significantly, but not completely, attenuated mechanical allodynia. In addition, the DC lesion before peripheral nerve injury did not prevent the generation of mechanical allodynia, which was completely blocked by subsequent contralateral hemisection of the spinal cord. However, unlike mechanical allodynia, DC lesion did not change thermal allodynia. These results suggest that the signals for mechanical allodynia following peripheral nerve injury are transmitted via the ipsilateral DC and the contralateral pathway(s).     

VPL-DBS on neuropathic pain rat model is effective in mechanical allodynia than cold allodynia

Recently, deep brain stimulation (DBS) is widely used in various types of neurodegenerative disorders for minimal invasiveness and safety of the procedure. Deep brain stimulation is consistently applied for the treatment of patients with neuropathic pain even though the success rate is not as high as other neurodegenerative disorders. Furthermore, it is also unclear how DBS improves neuropathic pain. In this study, we investigated the role of DBS following the stimulation parameter for analgesic effect on mechanical allodynia and cold allodynia in neuropathic pain rats. We used a sciatic nerve injury model to induce neuropathic pain, and observed responses to mechanical and cold stimulation by the von Frey test and acetone test, respectively. We classified the rats into four groups: na?ve (na?ve, n?=?10), na?ve?+?DBS (N?+?DBS, n?=?10), neuropathic pain (NP, n?=?10), and neuropathic pain?+?DBS (NP?+?DBS, n?=?10). We inserted the DBS electrode into the ventral posterolateral nucleus (VPL) into the rats (VPL-DBS). The score for mechanical allodynia was significantly decreased in NP?+?DBS group (p?<?0.01). However, the score for cold allodynia did not significantly drop in any groups including NP?+?DBS group (p?>?0.05). In this study, we found that the electrical stimulation of the VPL works more effectively with mechanical allodynia than cold one, and pain signal induced by mechanical stimulus and cold stimulus may be processed through different pathways in the brain.     

Effects of morphine on mechanical allodynia in a rat model of central neuropathic pain

We examined whether morphine reduced the behavioral signs of neuropathic pain below level induced by T13 spinal hemisection in rats. In order to examine the effect of morphine on the mechanical allodynia, morphine alone, morphine with naloxone and saline were administered intraperitoneally and intrathecally and behavioral tests were conducted. In systemic injection, mechanical allodynia was reduced only when a higher concentration of morphine (5 mg/kg) was used. Intrathecally injected morphine (0.5, 1, 2, 5 microg) reduced mechanical allodynia dose-dependently. It is suggested that systemic morphine has limited effect on mechanical allodynia but direct spinal administration of morphine is more effective in controlling central pain following spinal cord injury.     

Anti-fibroblast growth factor-2 antibodies attenuate mechanical allodynia in a rat model of neuropathic pain

Peripheral nerve injury leads to the activation of spinal cord astrocytes, which contribute to maintaining neuropathic (NP) pain behavior. Fibroblast growth factor-2 (FGF-2), a neurotrophic and gliogenic factor, is upregulated by spinal cord astrocytes in response to ligation of spinal nerves L5 and L6 (spinal nerve ligation [SpNL]). To evaluate the contribution of spinal astroglial FGF-2 to mechanical allodynia following SpNL, neutralizing antibodies to FGF-2 were injected intrathecally. Administration of 18 microg of anti-FGF-2 antibodies attenuated mechanical allodynia at day 21 after SpNL and reduced FGF-2 and glial acidic fibrillary protein mRNA expression and immunoreactivity in the L5 spinal cord segment of rats with SpNL. These results suggest that endogenous astroglial FGF-2 contributes to maintaining NP tactile allodynia associated with reactivity of spinal cord astrocytes and that inhibition of spinal FGF-2 ameliorates NP pain signs.     

Anti-fibroblast growth factor-2 antibodies attenuate mechanical allodynia in a rat model of neuropathic pain

Peripheral nerve injury leads to the activation of spinal cord astrocytes, which contribute to maintaining neuropathic (NP) pain behavior. Fibroblast growth factor-2 (FGF-2), a neurotrophic and gliogenic factor, is upregulated by spinal cord astrocytes in response to ligation of spinal nerves L5 and L6 (spinal nerve ligation [SpNL]). To evaluate the contribution of spinal astroglial FGF-2 to mechanical allodynia following SpNL, neutralizing antibodies to FGF-2 were injected intrathecally. Administration of 18 μg of anti-FGF-2 antibodies attenuated mechanical allodynia at day 21 after SpNL and reduced FGF-2 and glial acidic fibrillary protein mRNA expression and immunoreactivity in the L5 spinal cord segment of rats with SpNL. These results suggest that endogenous astroglial FGF-2 contributes to maintaining NP tactile allodynia associated with reactivity of spinal cord astrocytes and that inhibition of spinal FGF-2 ameliorates NP pain signs.     

Effects of electroacupuncture on cold allodynia in a rat model of neuropathic pain: mediation by spinal adrenergic and serotonergic receptors

The present study was performed to examine the effects of electroacupuncture (EA) on cold allodynia and its mechanisms related to the spinal adrenergic and serotonergic systems in a rat model of neuropathic pain. For the neuropathic surgery, the right superior caudal trunk was resected at the level between S1 and S2 spinal nerves innervating the tail. Two weeks after the nerve injury, EA stimulation (2 or 100 Hz) was delivered to Zusanli (ST36) for 30 min. The behavioral signs of cold allodynia were evaluated by the tail immersion test [i.e., immersing the tail in cold water (4 degrees C) and measuring the latency to an abrupt tail movement] before and after the stimulation. And then, we examined the effects of intrathecal injection of prazosin (alpha1-adrenoceptor antagonist, 30 microg), yohimbine (alpha2-adrenoceptor antagonist, 30 microg), NAN-190 (5-HT1A antagonist, 15 microg), ketanserin (5-HT2A antagonist, 30 microg), and MDL-72222 (5-HT3 antagonist, 12 microg) on the action of EA stimulation. Although both 2 Hz and 100 Hz EA significantly relieved the cold allodynia signs, 2 Hz EA induced more robust effects than 100 Hz EA. In addition, intrathecal injection of yohimbine, NAN-190, and MDL-72222, but not prazosin and ketanserin, significantly blocked the relieving effects of 2 Hz EA on cold allodynia. These results suggest that low-frequency (2 Hz) EA is more suitable for the treatment of cold allodynia than high-frequency (100 Hz) EA, and spinal alpha2-adrenergic, 5-HT1A and 5-HT3, but not alpha1-adrenergic and 5-HT2A, receptors play important roles in mediating the relieving effects of 2 Hz EA on cold allodynia in neuropathic rats.     

Intrathecal anti-IL-6 antibody and IgG attenuates peripheral nerve injury-induced mechanical allodynia in the rat: possible immune modulation in neuropathic pain

Interleukin-6 (IL-6) is a pleiotrophic cytokine with a diverse range of actions including the modulation of the peripheral and central nervous system. We have previously shown significant IL-6 protein and messenger RNA elevation in rat spinal cord following peripheral nerve injury that results in pain behaviors suggestive of neuropathic pain. These spinal IL-6 levels correlated directly with the mechanical allodynia intensity following nerve injury. In the current study, we sought to determine whether it is possible to attenuate mechanical allodynia and/or alter spinal glial activation resulting from peripheral nerve injury by specific manipulation of IL-6 with neutralizing antibodies or by global immune modulation utilizing immunogamma-globulin (IgG). Effects of peripheral administration of normal goat IgG and intrathecal (i.t.) administration of IL-6 neutralizing antibody, normal goat or normal rat IgG on mechanical allodynia associated with L5 spinal nerve transection were compared. Spinal glial activation was assessed at day 10 post surgery by immunohistochemistry. Low dose (0.01-0.001 microg) goat anti-rat IL-6 i.t. administration (P=0.025) significantly decreased allodynia and trended towards significance at the higher dose (0.08 microg to 0.008 microg, P=0.062). Low doses (0.01-0.001 microg) i.t. normal goat and rat IgG significantly attenuated mechanical allodynia, but not at higher doses (0.08-0.008 microg; P=0.001 for both goat and rat IgG). Peripherally administered normal goat IgG (30 or 100 mg/kg) did not attenuate mechanical allodynia. Spinal glial activation was unaltered by any treatment. These data provide further evidence for the role of central IL-6 and neuroimmune modulation in the etiology of mechanical allodynia following peripheral nerve injury.     

Relieving effects of electroacupuncture on mechanical allodynia in neuropathic pain model of inferior caudal trunk injury in rat: mediation by spinal opioid receptors

The relieving effects of electroacupuncture (EA) on mechanical allodynia and its mechanism related to the spinal opioid system were investigated in a rat model of neuropathic pain. To produce neuropathic pain in the tail, the right superior caudal trunk was resected between the S1 and S2 spinal nerves. Two weeks after the surgery, EA stimulation (2 or 100 Hz, 0.3 ms, 0.2-0.3 mA) was delivered to Zusanli (ST36) for 30 min. The degree of mechanical allodynia was evaluated quantitatively by touching the tail with von Frey hair (2.0 g) at 10 min intervals. These rats were then subjected to an i.t. injection with one of the three specific opioid agonists in successive ways: the mu agonist (DAMGO 25, 50 and 100 pmol), the delta agonist (DADELT II 0.5, 1 and 2 nmol), and the kappa agonist (U50488H 5, 10 and 20 nmol) separated by 10 min in cumulative doses. During 30 min of EA stimulation, specific opioid antagonists were subjected to i.t. injection: the mu antagonist (beta-FNA 5, 10 and 20 nmol), the delta antagonist (naltrindole 5, 10 and 20 nmol), and the kappa antagonist (nor-BNI 3, 6 and 12 nmol) separated by 10 min in cumulative doses. As a result, EA reduced the behavioral signs of mechanical allodynia. Two Hz EA induced a robust and longer lasting effect than 100 Hz. All three opioid agonists also showed relieving effects on mechanical allodynia. However, nor-BNI could not block the EA effects on mechanical allodynia, whereas beta-FNA or naltrindole significantly blocked EA effects. These results suggest that the mu and delta, but not kappa, opioid receptors in the spinal cord of the rat, play important roles in mediating relieving effects on mechanical allodynia induced by 2 Hz EA.     

Evidence for suppression of electroacupuncture on spinal glial activation and behavioral hypersensitivity in a rat model of monoarthritis

Our previous study demonstrated that single intrathecal (i.t.) application of fluorocitrate, a glial metabolic inhibitor, synergized electroacupuncture (EA) antagonizing behavioral hypersensitivity in complete Freund's adjuvant (CFA)-induced monoarthritic rat. To further investigate the relationship between spinal glial activation and EA analgesia, the present study examined the effects of multiple EA on spinal glial activation evoked by monoarthritis (MA). The results showed that (1) unilateral intra-articular injection of CFA produced a robust glial activation on the spinal cord, which was associated with the development and maintenance of behavioral hypersensitivity; (2) multiple EA stimulation of ipsilateral "Huantiao" (GB30) and "Yanglingquan" (GB34) acupoints or i.t. injection of fluorocitrate (1 nmol) significantly suppressed spinal glial activation; (3) inhibitory effects of EA on spinal glial activation and behavioral hypersensitivity were significantly enhanced when EA combined with fluorocitrate, indicating that disruption of glial function may potentiate EA analgesia in inflammatory pain states. These data suggested that analgesic effects of EA might be associated with its counter-regulation to spinal glial activation, and thereby provide a potential strategy for the treatment of arthritis.     

Glatiramer acetate attenuates neuropathic allodynia through modulation of adaptive immune cells

Immune-neuronal interactions contribute to neuropathic pain. Thus, immune-competent cells such as microglia may provide targets for pain relief, as may infiltrating lymphocytes. We evaluated the nature of the lymphocyte response in the spinal cord in association with the maintenance of neuropathic allodynia. We assessed T cell contribution to pain processing by targeting these cells with Glatiramer acetate (GA) which when administered systemically reversed neuropathic allodynia, inhibited microglia response and increased IL-10 and IL-4 expressing T cells in neuropathic dorsal horns. These studies advance understanding of lymphocyte contribution to chronic pain and reveal a new mechanism of T cell intervention.     

Intrathecal transplantation of neuroblastoma cells decreases heat hyperalgesia and cold allodynia in a rat model of neuropathic pain

Intrathecal grafting of cells as biological pumps to deliver monoamines, endorphins, and/or trophic factors, has been shown to be effective in treating chronic pain both in experimental animals and in clinical trials. We have tested whether intrathecal implantation of neuroblastoma cells reduces heat hyperalgesia and cold allodynia in a rat model of neuropathic pain induced by chronic constriction injury (CCI) of the sciatic nerve. Behavioral tests and cerebrospinal fluid (CSF) collection were performed before CCI, 1 week later (after which, vehicle or NB69 cells were intrathecally injected) and at 4, 7, and 14 days post-injection. Both CSF sampling and injection of the cells were performed by direct lumbar puncture. Intrathecal grafting of 4 x 10(6) NB69 neuroblastoma cells reduced to basal levels the nociceptive response to heat in nerve-injured hindpaws, while the response of control limbs remained unchanged. Similarly, the allodynic response to cold elicited by acetone evaporation decreased in the animals implanted with NB69 cells. An increase in the concentrations of dopamine and serotonin metabolites of around 150% was observed in the CSF of animals that received grafts of NB69 cells. These data suggest that the monoamines released by NB69 cells in the intrathecal space produce analgesia to neuropathic pain in rats.     

Effects of glial cell line-derived neurotrophic factor intrathecal injection on spinal dorsal horn glial fibrillary acidic protein expression in a rat model of neuropathic pain

Glial fibrillary acidic protein (GFAP) is a specific astrocytic marker in the central nervous system. Few studies on the effects of glial cell line-derived neurotrophic factor (GDNF) intrathecal injection on GFAP expression exist in the literature. The present study determined GFAP expression in rat spinal dorsal horn following a spinal nerve ligation (SNL). The effects of GDNF intrathecal injection on GFAP expression were examined to gather experimental evidence on the mechanisms underlying neuropathic pain. Following L5-6 SNL, male Sprague-Dawley rats were randomly divided into four groups: normal control, sham-operated, SNL, and GDNF. Each group was further divided into three subgroups (n = 10) according to the times of sacrifice: 3, 7, and 14 days after surgery. Compared with the normal control and the sham-operated groups, GFAP expression in the SNL group increased at day 3 after surgery and lasted until 14 days after. GFAP expression was significantly less in the GDNF group compared with the SNL group which lasted until 14 days after surgery, suggesting that rat spinal dorsal horn GFAP expression contributes to SNL-induced neuropathic pain. The mechanisms underlying GDNF alleviation of neuropathic pain were shown to be related to the GDNF inhibition of GFAP expression in the spinal dorsal horn.     

Morphine amplifies mechanical allodynia via TLR4 in a rat model of spinal cord injury

Central neuropathic pain (CNP) is a pervasive, debilitating problem that impacts thousands of people living with central nervous system disorders, including spinal cord injury (SCI). Current therapies for treating this type of pain are ineffective and often have dose-limiting side effects. Although opioids are one of the most commonly used CNP treatments, recent animal literature has indicated that administering opioids shortly after a traumatic injury can actually have deleterious effects on long-term health and recovery. In order to study the deleterious effects of administering morphine shortly after trauma, we employed our low thoracic (T13) dorsal root avulsion model (Spinal Neuropathic Avulsion Pain, SNAP). Administering a weeklong course of 10 mg/kg/day morphine beginning 24 h after SNAP resulted in amplified mechanical allodynia. Co-administering the non-opioid toll-like receptor 4 (TLR4) antagonist (+)-naltrexone throughout the morphine regimen prevented morphine-induced amplification of SNAP. Exploration of changes induced by early post-trauma morphine revealed that this elevated gene expression of TLR4, TNF, IL-1β, and NLRP3, as well as IL-1β protein at the site of spinal cord injury. These data suggest that a short course of morphine administered early after spinal trauma can exacerbate CNP in the long term. TLR4 initiates this phenomenon and, as such, may be potential therapeutic targets for preventing the deleterious effects of administering opioids after traumatic injury.     

The effect of perineural colchicine on nerve injury-induced spinal glial activation and neuropathic pain behavior.

Factors transported centrally from the site of a peripheral nerve injury are known to provide cellular activation signals to the dorsal root ganglion and spinal cord. Yamamoto and Yaksh [35] were able to use colchicine disruption of axonal transport to abolish thermal hyperalgesia after sciatic chronic constriction in the rat. The current study set out to ascertain whether this observation could be reproduced by applying the same pharmacologic paradigm to a complete, segmentally specific, spinal nerve tight ligation (SPTL) and assessing the impact of this treatment on mechanical allodynia and central, spinal glial activation. Mechanical allodynia of the ipsilateral (lesion side) hind paw was measured at 1, 3, 5, 7, 10, and 14 days following SPTL. Spinal astrocytic and microglial activation were assessed immunohistochemically at 5 and 14 days. Colchicine was unable to prevent mechanical allodynia or spinal glial activation when applied perineurally just proximal to the site of SPTL. Administered alone, colchicine (without SPTL) induced both astrocytic and microglial activation, but not mechanical allodynia. Colchicine applied distal to the site of SPTL did not alter mechanical allodynia or glial responses to SPTL. Neuronal tracing experiments were performed to verify segmental disruption of axonal transport by either SPTL or colchicine treatment. Neuronal tracer injected into the sciatic nerve could not be found at the L5 spinal level following perineural colchicine treatment or tight ligation of the L5 spinal nerve, however, tracer was present at the unobstructed L4 spinal level. These results suggest that central astrocytic and microglial responses may be triggered by disruption of transported signals from the periphery, because they are induced by either colchicine or tight ligation. Conversely, axonally transported factors, either from the site of nerve injury or from the periphery, do not appear to be critical for the development of mechanical allodynia.     

AIDA reduces glutamate release and attenuates mechanical allodynia after spinal cord injury

Spinal cord injury (SCI) leads to an increase in extracellular excitatory amino acid (EAA) concentrations, resulting in glutamate receptor-mediated excitotoxicity and central sensitization. To test contributions of group I metabotropic glutamate receptors (mGluRs) in SCI induced release of glutamate and in behavioral outcomes of central sensitization following injury, we administered 1-aminoindan-1,5-dicarboxylic acid (AIDA; 0.1 nmol intraspinally), a potent group I mGluR antagonist, to rats immediately after spinal cord contusion injury. EAAs were collected by microdialysis and quantified using HPLC. AIDA significantly decreased extracellular glutamate but not aspartate concentrations and significantly attenuated the development of mechanical but not thermal allodynia. These results suggest mGluRs play an important role in injury-induced EAA release and in central sensitization following SCI.     

Neuronal NR2B-containing NMDA receptor mediates spinal astrocytic c-Jun N-terminal kinase activation in a rat model of neuropathic pain

Spinal N-methyl d-aspartate receptor (NMDAR) plays a pivotal role in nerve injury-induced central sensitization. Recent studies suggest that NMDAR also contributes to neuron-astrocyte signaling. c-Jun N-terminal kinase (JNK) is persistently and specifically activated (indicated by phosphorylation) in spinal cord astrocytes after nerve injury and thus it is considered as a dependable indicator of pain-related astrocytic activation. NMDAR-mediated JNK activation in spinal dorsal horn might be an important form of neuron-astrocyte signaling in neuropathic pain. In the present study, we observed that intrathecal injection of MK-801, a noncompetitive NMDA receptor antagonist, or Ro25-6981 and ifenprodil, which are selective antagonists of NR2B-containing NMDAR each significantly reduced nerve injury-induced JNK activation. Double immunostaining showed that NR2B was highly expressed in neurons, indicating the effect of NMDAR antagonists on JNK activation was indirect. We further observed that intrathecal injection of NMDA (twice a day for 3 days) significantly increased spinal JNK phosphorylation. Besides, NMDAR-related JNK activation could be blocked by a neuronal nitric oxide synthase (nNOS) selective inhibitor (7-nitroindazole sodium salt) but not by a nNOS sensitive guanylyl cyclase inhibitor (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one). Finally, real-time RT-PCR and immunostaining showed that nerve injury-induced interleukin-1beta expression was dependent on astrocytic JNK activation. Treatments targeting NMDAR-nNOS pathway also influenced interleukin-1beta expression, which further confirmed our hypothesis. Taken together, our results suggest that neuronal NMDAR-nNOS pathway could activate astrocytic JNK pathway. Excitatory neuronal transmission initiates astrocytic activation-induced neuroinflammation in this way, which contributes to nerve injury-induced neuropathic pain.     

Spatial and temporal activation of spinal glial cells: role of gliopathy in central neuropathic pain following spinal cord injury in rats

In the spinal cord, neuron and glial cells actively interact and contribute to neurofunction. Surprisingly, both cell types have similar receptors, transporters and ion channels and also produce similar neurotransmitters and cytokines. The neuroanatomical and neurochemical similarities work synergistically to maintain physiological homeostasis in the normal spinal cord. However, in trauma or disease states, spinal glia become activated, dorsal horn neurons become hyperexcitable contributing to sensitized neuronal-glial circuits. The maladaptive spinal circuits directly affect synaptic excitability, including activation of intracellular downstream cascades that result in enhanced evoked and spontaneous activity in dorsal horn neurons with the result that abnormal pain syndromes develop. Recent literature reported that spinal cord injury produces glial activation in the dorsal horn; however, the majority of glial activation studies after SCI have focused on transient and/or acute time points, from a few hours to 1 month, and peri-lesion sites, a few millimeters rostral and caudal to the lesion site. In addition, thoracic spinal cord injury produces activation of astrocytes and microglia that contributes to dorsal horn neuronal hyperexcitability and central neuropathic pain in above-level, at-level and below-level segments remote from the lesion in the spinal cord. The cellular and molecular events of glial activation are not simple events, rather they are the consequence of a combination of several neurochemical and neurophysiological changes following SCI. The ionic imbalances, neuroinflammation and alterations of cell cycle proteins after SCI are predominant components for neuroanatomical and neurochemical changes that result in glial activation. More importantly, SCI induced release of glutamate, proinflammatory cytokines, ATP, reactive oxygen species (ROS) and neurotrophic factors trigger activation of postsynaptic neuron and glial cells via their own receptors and channels that, in turn, contribute to neuronal-neuronal and neuronal-glial interaction as well as microglia-astrocytic interactions. However, a systematic review of temporal and spatial glial activation following SCI has not been done. In this review, we describe time and regional dependence of glial activation and describe activation mechanisms in various SCI models in rats. These data are placed in the broader context of glial activation mechanisms and chronic pain states. Our work in the context of work by others in SCI models demonstrates that dysfunctional glia, a condition called "gliopathy", is a key contributor in the underlying cellular mechanisms contributing to neuropathic pain.     

A Chronic fatigue syndrome model demonstrates mechanical allodynia and muscular hyperalgesia via spinal microglial activation

Patients with chronic fatigue syndrome (CFS) and fibromyalgia syndrome (FMS) display multiple symptoms, such as chronic widespread pain, fatigue, sleep disturbance, and cognitive dysfunction. Abnormal pain sensation may be the most serious of these symptoms; however, its pathophysiology remains unknown. To provide insights into the molecular basis underlying abnormal pain in CFS and FMS, we used a multiple continuous stress (CS) model in rats, which were housed in a cage with a low level of water (1.5 cm in depth). The von Frey and Randall–Seritto tests were used to evaluate pain levels. Results showed that mechanical allodynia at plantar skin and mechanical hyperalgesia at the anterior tibialis (i.e., muscle pain) were induced by CS loading. Moreover, no signs of inflammation and injury incidents were observed in both the plantar skin and leg muscles. However, microglial accumulation and activation were observed in L4–L6 dorsal horn of CS rats. Quantification analysis revealed a higher accumulation of microglia in the medial part of Layers I–IV of the dorsal horn. To evaluate an implication of microglia in pain, minocycline was intrathecally administrated (via an osmotic pump). Minocycline significantly attenuated CS‐induced mechanical hyperalgesia and allodynia. These results indicated that activated microglia were involved in the development of abnormal pain in CS animals, suggesting that the pain observed in CFS and FMS patients may be partly caused by a mechanism in which microglial activation is involved. GLIA 2014;62:1407–1417