Development Of Inflammatory Pain Research Articles

Expression of acetylated histone 3 in the spinal cord and the effect of morphine on inflammatory pain in rats.

In this study, a rat model of inflammatory pain was produced by injecting complete Freund's adjuvant into the hind paw, and the expression of acetylated histone 3 in the spinal cord dorsal horn was examined using immunohistochemical staining. One day following injection, there was a dramatic decrease in acetylated histone 3 expression in spinal cord dorsal horn neurons. However, on day 7, expression recovered in adjuvant-injected rats. While acetylated histone 3 labeling was present in dorsal horn neurons, it was more abundant in astrocytes and microglial cells. The recovery of acetylated histone 3 expression was associated with a shift in expression of the protein from neurons to glial cells. Morphine injection significantly upregulated the expression of acetylated histone 3 in spinal cord dorsal horn neurons and glial cells 1 day after injection, especially in astrocytes, preventing the transient downregulation. Our results indicate that inflammatory pain induces a transient downregulation of acetylated histone 3 in the spinal cord dorsal horn at an early stage following adjuvant injection, and that this effect can be reversed by morphine. Thus, the downregulation of acetylated histone 3 may be involved in the development of inflammatory pain.

Phosphoinositide Regulation of TRPV1

TRPV1 is a nonselective calcium permeable cation channel present in polymodal nociceptors that plays a crucial role in the development of inflammatory pain and thermal hypersensitivity. Plasmamembrane phosphoinositides are recognized as important regulators of TRPV1; the precise nature of their effect is, however, controversial. We and others have shown that phosphatidyl-inositol-(4,5)-bisphosphate [PI(4,5)P2] as well as other phosphoinositides activate TRPV1 in excised patches. Calcium influx via TRPV1 channels activates PLC and results in a robust depletion of both phosphatidyl-inositol-(4)- phosphate [PI(4)P] and PI(4,5)P2 in expression systems. Hydrolysis of PI(4,5)P2 under these circumstances is now accepted to be an important contributor to channel desensitization. However, the involvement of PI(4)P in the process remains an issue of debate. In addition, preceding data corroborated by our own observations suggest an additional indirect inhibitory effect of PI(4,5)P2, but not other phosphoinositides. In the present work we show that potentiation of whole-cell TRPV1 currents by bradykinin is partially impaired by dialyzing diC8-PI(4,5)P2 through the patch pipette in both expression systems and native cells (DRG neurons), supporting the notion that in addition to the well documented role of PKC-mediated phosphorylation in TRPV1 channel sensitization, PI(4,5)P2 depletion may also be involved in this phenomenon. Using mouse DRG neurons we confirm that in addition to PI(4,5)P2, PI(4)P is also an important activator of TRPV1. Both lipids are simultaneously depleted in response to TRPV1-activation, while dialysis of both lipids via the patch pipette reduced desensitization of TRPV1-positive neurons. In contrast Bradykinin receptor activation differentially regulated PI(4,5)P2 and PI(4)P abundance in DRG neurons, resulting in isolated PI(4,5)P2 depletion. These observations suggest important differences in phosphoinositides handling during calcium-activated and receptor-induced PLC activation, respectively, and may partially explain the differential TRPV1 regulation under these conditions.

Role of peripheral polyamines in the development of inflammatory pain

Polyamines (putrescine, spermidine and spermine) are aliphatic amines that are produced by the action of ornithine decarboxylase (ODC) in a rate-limiting and protein kinase C (PKC)-regulated step. Because high levels of polyamines are found in the synovial fluid of arthritic patients, the aim of the present study was to identify the role of peripherally produced polyamines in a model of inflammatory pain induced by adjuvant. The subcutaneous injection of Complete Freund's adjuvant (CFA, 50μL/paw) caused the development of mechanical allodynia and edema. Moreover, it increased ODC expression and activity and PKC activation. Administration of the selective ODC inhibitor DFMO (10μmol/paw) attenuated the development of allodynia and edema and decreased ODC activity in both control and CFA-treated animals. Furthermore, administration of the PKC inhibitor GF109203X (1nmol/paw) reduced allodynia and ODC activity in animals injected with CFA. A subcutaneous injection of putrescine (10μmol/paw), spermidine (3–10μmol/paw) or spermine (0.3–3μmol/paw) into the rat paw also caused mechanical allodynia and edema. The present results suggest that endogenously synthesized polyamines are involved in the development of nociception and edema caused by an adjuvant. Moreover, polyamine production in inflammatory sites seems to be related to an increase in ODC activity stimulated by PKC activation. Thus, controlling polyamine synthesis and action could be a method of controlling inflammatory pain.

Complex Regulation of TRPV1 by Phosphoinositides

TRPV1 is a nonselective calcium permeable cation channel present in polymodal nociceptors that plays a crucial role in the development of inflammatory pain and thermal hypersensitivity. Plasmamembrane phosphoinositides are recognized as important regulators of TRPV1; the precise nature of their effect is, however, controversial. Recent studies indicate that phosphatidyl inositol (4,5) bisphosphate [PI(4,5)P2] and other phosphoinositides activate TRPV1 in excised patches. In concert with previous studies, we have shown that calcium influx-mediated activation of PLC results in a robust depletion of both phosphatidyl inositol (4) phosphate [PI(4)P] and PI(4,5)P2 and is one of the mechanisms of TRPV1 desensitization.

Substance P in the cerebrospinal fluid-contacting nucleus contributes to morphine physical dependence in rats

The cerebrospinal fluid-contacting nucleus (CSF-CN), distributes and localizes in the ventral periaqueductal central gray (PAG) of the brainstem, which may influence actual composition of the cerebrospinal fluid (CSF) for non-synaptic signal transmission via releasing or absorbing bioactive substances. Many experiments have demonstrated that substance P (SP), a substance that is shown to be up-regulated in CSF-CN, plays an important role in the development of inflammatory pain and neuropathic pain. Thus in the present study, we hypothesize that SP in CSF-CN might contribute to morphine dependence in rats, inhibiting SP with (D-Pro2, D-Phe7, D-Trp9)-SP intracerebroventricular (i.c.v.) injection reduce chronic morphine dependence and withdrawal. Rats were repeatedly injected with morphine in five escalating doses for morphine physical dependence. Morphine withdrawal-like behavioral signs and morphine analgesia behaviors were monitored after naloxone administration following i.c.v. injection of (D-Pro2, D-Phe7, D-Trp9)-SP. And SP-expression of CSF-CN was evaluated with dual-label immunofluorescent technique on morphine withdrawal in rats. After i.c.v. treatment with (D-Pro2, D-Phe7, D-Trp9)-SP, the naloxone-precipitated withdrawal symptoms were significantly attenuated, paw withdrawal threshold/thermal withdrawal latency (PWT/TWL) were increased, and SP-expression in CSF-CN was significantly reduced than control group. SP, known a neurotransmitter/neuromodulator of nociception, has also been implicated in the signs of opioid withdrawal. This study provides the first evidence that SP in CSF-CN contributes to morphine physical dependence and withdrawal, which may provide an important and specific role in mediating the motivational aspects of opiates withdrawal via CSF – the parenchyma of the brain, and may represent a novel pharmacological route such as SP inhibitor i.c.v. injection for the control of drug abuse.

Role of gliocytes in the spinal cord in development of inflammatory pain in rats

Objective To evaluate the role of gliocytes in the spinal cord in the development of inflammatory pain (IP) in rats. Methods Adult male SD rats weighing 180-220 g were used in this experiment. A catheter was implanted in the subarachnoid space according to the method described by Yang. Animals with abnormal motor function of the hindlimb after intrathecal (IT) catheter implantation were excluded. IP was induced by subcutaneous (sc) injection of complete Freund's adjuvant (CFA) 50 μl at the lateral side of the ankle joint of the right hindpaw. Sixty-five rats were randomly divided into 5 groups ( n = 13 each): group I IP control normal saline (NS) 50μl was injected sc instead of CFA; group II IP; group IE PC (IT) + IP control fluorinated citric acid (FC, a gliocyte metabolism inhibitor) 1 nmol/10μl was injected IT at 15 min before NS 50 μl sc injection; group IV NS (IT) + IP and group V FC (IT) + IP. The mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured 2 d before induction of IP (T_0, baseline) .before and at 2, 4, 6, 8, 10, 12, 24 and 26 h (T_(1-9)) after sc NS or CFA injection. Five enimals in each group were killed at T_5 (8 h after sc NS/CFA injection) and the lumbar segment (L_(4,5)) was removed for determination of glial fibrillary acidic protein ( CFAP) and OX-42 expression by immuno-histochemistry. Results In group Ⅱ and Ⅳ sc CFA significantly decreased MWT and TWL. Mechanical and thermal hyperalgegia induced by sc CFA was significantly suppressed by intrathecal FC in group V . IP significantly increased GFAP and OX-42 expression in the spinal cord. Intrathecal FC significantly attenuated IP-induced up-regulation of GFAP and OX-42 expression in the spinal cord. Conclusion The activation of gliocytes in the spinal cord is involved in the development of CFA-induced hyperalgesia in rats. Key words: Astrocytes; Microglia; Pain; Spinal cord

Complex Regulation of TRPV1 by Phosphoinositides

TRPV1 is a nonselective highly calcium permeable cation channel present in the peripheral nervous system exclusively in polymodal nociceptors. A sensory integrator of several noxious stimuli, TRPV1 plays a crucial role in the development of inflammatory pain and hypersensitivity. Plasmamembrane phosphoinositides are recognized as important regulators of TRPV1 function; the precise nature of their effect is, however, controversial.Phosphatidylinositol-4,5-bisphosphate (PIP2 ) has initially been proposed to tonally inhibit TRPV1 via a C-terminal inhibitory domain. Furthermore, receptor-mediated depletion of PIP2 was proposed to be involved in sensitization of TRPV1 in response to pro-inflammatory agents. However, in subsequent studies including our own, direct intracellular application of PIP2 reproducibly potentiated TRPV1 currents rather than inhibiting them. In addition, PIP2 depletion concurrent with robust TRPV1 activation is an important contributing factor to channel desensitization consistent with the activating effect of PIP2. We attempt to address this controversy utilizing multiple independent approaches to selectively regulate plasmamembrane PIP2 levels in heterologous expression systems. Our results show that TRPV1 currents in intact cells elicited by low to moderate, but not high agonist concentrations are potentiated in response to PIP2 depletion. Conversely, increasing PIP2 levels inhibits low but not high agonist-induced TRPV1 currents. These effects are reduced or absent in the mutant channel lacking the putative C-terminal inhibitory domain. The inhibitory effect of PIP2 however was never observed in excised patches even at low agonist concentrations. Our results are consistent with an agonist concentration-dependent dual regulatory effect of PIP2. The inhibitory effect furthermore appears to be indirect. Such dual effects of PIP2 have previously been described for voltage-gated calcium channels as well as other TRP channels and raise important questions as to the identity of interacting molecules conferring the inhibitory effect and the physiological relevance of such complex regulation.

Activity-dependent potentiation of calcium signals in spinal sensory networks in inflammatory pain states

The second messenger calcium is a key mediator of activity-dependent neural plasticity. How persistent nociceptive activity alters calcium influx and release in the spinal cord is not well-understood. We performed calcium-imaging on individual cell bodies and the whole area within laminae I and II in spinal cord slices from mice in the naïve state or 24 h following unilateral hindpaw plantar injection of complete Freund’s adjuvant. Calcium signals evoked by dorsal root stimulation at varying strengths displayed a steep rise and slow decay over 15–20 s and increased progressively with both increasing intensity and frequency of stimulation in naïve mice. Experiments with pharmacological inhibitors revealed that both ionotropic glutamate receptors and intracellular calcium stores contributed to maximal calcium signals in laminae I and II evoked by stimulating dorsal roots at 100 Hz frequency. Importantly, as compared to naïve mice, we observed that in mice with unilateral hindpaw inflammation, calcium signals were potentiated to 159 ± 10% in the ipsilateral dorsal horn and 179 ± 8% in the contralateral dorsal horn. In addition to the contribution from NMDA receptors, GluR-A-containing AMPA receptors were found to be critically required for the above changes in spinal calcium signals, as revealed by analysis of genetically modified mouse mutants, whereas intracellular calcium release was not required. Thus, these results suggest that there is an important functional link between calcium signaling in superficial spinal laminae and the development of inflammatory pain. Furthermore, they highlight the importance of GluR-A-containing calcium-permeable AMPA receptors in activity-dependent plasticity in the spinal cord.

Topical ketanserin attenuates hyperalgesia and inflammation in arthritis in rats

We investigated effects of topical application of ketanserin, a 5-HT 2A receptor antagonist, on hyperalgesia and edema in the arthritic rat, a chronic pain model with inflammation. Unilateral, but not bilateral, arthritis was induced with intra-articular injection of a mixture of kaolin and carrageenan in one side, as indicated by the shortened paw withdrawal latency and an increase in the circumference of the knee joint. Topical application of ketanserin onto skin over the arthritic joint delivered in a mixture of gelatin, glycerol and kaolin produced dose-dependent attenuation of nociceptive and inflammatory effects resulting from intra-articularly injected kaolin/carrageenan. One and 3% ketanserin produced significant or even complete anti-hyperalgesia, as well as a remarkable anti-inflammatory effect (50–70% reduction of edema) while 0.3% ketanserin and placebo failed to produce any effect. Moreover, the effects of ketanserin were maintained for 13 days without decline. In contrast, 3% ketanserin applied to skin of the knee joint on the non-inflamed side for 2 weeks did not alter nociceptive thresholds of the paw and the size of the knee joint in both the inflamed and non-inflamed limbs. These results indicate that 5-HT 2A receptors in the periphery play a significant role in the maintenance and/or development of inflammatory pain. The present study suggests that topical ketanserin is a promising direction for potential clinical exploration to relieve established hyperalgesia and inflammation in arthritis without adverse effects and tolerance.

Neuropathic Pain Develops Normally in Mice Lacking both Nav1.7 and Nav1.8

Two voltage gated sodium channel α-subunits, Nav1.7 and Nav1.8, are expressed at high levels in nociceptor terminals and have been implicated in the development of inflammatory pain. Mis-expression of voltage-gated sodium channels by damaged sensory neurons has also been implicated in the development of neuropathic pain, but the role of Nav1.7 and Nav1.8 is uncertain. Here we show that deleting Nav1.7 has no effect on the development of neuropathic pain. Double knockouts of both Nav1.7 and Nav1.8 also develop normal levels of neuropathic pain, despite a lack of inflammatory pain symptoms and altered mechanical and thermal acute pain thresholds. These studies demonstrate that, in contrast to the highly significant role for Nav1.7 in determining inflammatory pain thresholds, the development of neuropathic pain does not require the presence of either Nav1.7 or Nav1.8 alone or in combination.

Prevention by celecoxib of secondary hyperalgesia induced by formalin in rats

Administration of formalin in rat paws results in stimulation of nociceptive pathways, which leads to an increase in the excitability of neurons present in dorsal horn. This increased neuron excitability, described as central sensitization, may result in development of inflammatory pain at a distant site of injury application, known as secondary hyperalgesia. The aim of the present study was to verify whether formalin injection in rat paws would lead to secondary hyperalgesia development, as measured by the tail-flick test. We also aimed to investigate whether celecoxib, a specific cyclooxygenase 2 (COX-2) inhibitor, would affect secondary hyperalgesia. Formalin injected into the rat paws significantly reduced the latency for a flick response in the rat tail, which characterized development of secondary hyperalgesia. In addition, formalin-induced secondary hyperalgesia was locally prevented by pre-but not post-celecoxib treatment. However, celecoxib administered spinally inhibited formalin-induced secondary hyperalgesia, either administered previously or following formalin. In contrast, piroxicam, an unspecific COX inhibitor which displays an increased selectivity towards COX-1, only prevented secondary hyperalgesia to formalin at a high dose following spinal administration. Taken together, these results suggest that COX-2 plays an important role both in the central and in the peripheral nerve sensitization following formalin administration in rat paws. They also suggested that once central sensitization starts it can no longer be blocked by a specific COX-2 inhibitor administered locally. Notwithstanding, spinal administration of a specific COX-2 inhibitor still blocks ongoing sensitization and prevents maintenance of central sensitization.

Transient up-regulation of spinal cyclooxygenase-2 and neuronal nitric oxide synthase following surgical inflammation.

Surgery induces pain and hyperalgesia postoperatively. The products of cyclooxygenases and nitric oxide synthase (NOS) have been implicated in the development of inflammatory pain and hyperalgesia experimentally, and the use of drugs clinically that modify cyclooxygenase activity has been advocated in the management of perioperative pain. However, regulation of these enzymes following surgery has not been studied. Adult female sheep (n = 12) undergoing a midline laparotomy for collection of ova were used in this study. Lumbar and cervical spinal cord tissue was collected from animals euthanized 1 day and 6 or 7 days after surgery and processed for cyclooxygenase (cyclooxygenase-1 and cyclooxygenase-2), neuronal NOS mRNA expression using reverse-transcription polymerase chain reaction and hybridization. Tissues were also processed for NADPH-diaphorase staining and cyclooxygenase-1 and cyclooxygenase-2 protein expression by immunohistochemistry and Western blotting. No alteration in cyclooxygenase-1 or cyclooxygenase-2 mRNA or protein concentrations were detected in spinal cord by reverse-transcription polymerase chain reaction and Western blotting, respectively, at 1 day or 6 or 7 days after surgery. However, using techniques that localize mRNA and protein expression ( hybridization and immunohistochemistry, respectively), increases in cyclooxygenase-2 were identified in lamina V dorsal horn neurons in lumbar spinal cord 1 day after surgery. A significant increase in neuronal NOS mRNA was observed in lumbar spinal cord 1 day after surgery, localized to laminae I-II and lamina V neurons, which returned to baseline concentrations by 6 to 7 days. NADPH-diaphorase staining was significantly increased in laminae I-II in lumbar spinal cord 1 day after surgery but not after 6 to 7 days. Spinal cyclooxygenase and neuronal NOS pathways are differentially altered following surgical inflammation. The early and transient nature of these changes suggests that these enzymes are implicated in postoperative pain and hypersensitivity.

Progressive tactile hypersensitivity: an inflammation-induced incremental increase in the excitability of the spinal cord

Two established phenomena contribute to the generation of post-injury pain hypersensitivity: peripheral sensitization, an increase in transduction sensitivity of high threshold Aδ and C-fibre nociceptors, and central sensitization, an increase in excitability of neurones in the spinal cord triggered exclusively by C-fibre inputs. We now describe a novel phenomenon: progressive tactile hypersensitivity, which contributes to a cumulative allodynia during inflammation. Behavioural measurements in conscious intact animals showed that repeated light touch stimuli delivered at 5-min intervals to an inflamed paw, established 48 h earlier by an intra-plantar injection of complete Freund's adjuvant (CFA), resulted in a progressive reduction in the mechanical withdrawal threshold by more than 75%, from its already hypersensitive basal level. This hypersensitive state persisted for several hours after discontinuing the touch stimuli and did not occur in non-inflamed animals. To monitor nociceptive processing and the afferent fibres responsible, we also measured activity in posterior biceps femoris/semitendinosus flexor motor neurons. In non-inflamed decerebrate-spinal rats, the cutaneous mechanical threshold and pinch-evoked activity of these neurones are stable when tested repeatedly at 5-min intervals and are characterised by absent or small responses to low intensity mechanical stimuli or electrical activation of Aβ-fibres. In inflamed animals, the spontaneous activity, touch-, pinch- and Aβ-afferent-evoked responses of hamstring flexor motor neurones are significantly increased. The flexor reflex becomes, moreover, progressively more sensitized by repetition every 5 min, of standard mechanical stimuli (touch and pinch), that do not modify excitability in control non-inflamed animals. A cumulative increase in Aβ-afferent-evoked responses also occurs when the test stimulus only comprises stimulation of the sural nerve at Aβ strength (10 Hz, 10 sec), showing that Aβ-afferents have the capacity to produce progressive hypersensitivity. Progressive hypersensitivity, measured here as a progressive tactile allodynia after inflammation in either intact or decerebrate-spinal rats, with its gradual build-up and contribution from Aβ fibres, is very different from the central sensitization induced by C-fibre stimulation which is characterised by a peak increase in excitability soon after the conditioning input followed by a steady decrement to baseline levels. Progressive hypersensitivity is likely to be the consequence of an alteration in the function and phenotype of afferents innervating inflamed tissue and the pattern of excitation they produce in spinal neurones. The phenomenon may have an important role in the development of inflammatory pain and hypersensitivity.