Blair D. Grubb

Prostanoids synthesized by cyclo‐oxygenase isoforms in rat spinal cord and their contribution to the development of neuronal hyperexcitability

1 The responses of wide dynamic range spinal dorsal horn neurones to noxious mechanical stimulation of the ankle or knee joint were tested before and after spinal administration of the non‐selective cyclo‐oxygenase (COX) inhibitors, indomethacin and meclofenamic acid. Neither of these drugs altered the responses of these neurones to noxious mechanical stimulation. 2 Wind‐up of a spinal nociceptive reflex evoked by electrical stimulation of the sural nerve at C‐fibre strength was dose‐dependently inhibited by intravenous administration of indomethacin, a non‐selective COX inhibitor, and SC58125, a selective COX‐2 inhibitor. Intrathecal administration of indomethacin also reduced the wind‐up of this nociceptive reflex. 3 Western blot analysis of proteins extracted from normal rat spinal cord revealed the presence of both cyclo‐oxygenase (COX)‐1 and COX‐2 proteins. 4 Immunocytochemistry of sections of normal rat spinal cord with specific COX‐1 antiserum revealed little specific COX‐1‐like immunoreactivity in the grey matter. With the same antiserum, intense COX‐1‐like immunoreactivity was observed in the cytoplasm, nuclear membrane and axonal processes of small to medium sized (<1000 μm2) dorsal root ganglion (DRG) cell bodies. 5 Immunocytochemistry of sections of normal rat spinal cord incubated with specific COX‐2 antiserum showed intense COX‐2‐like immunoreactivity (COX‐2‐li) in the superficial dorsal horn of the spinal cord (laminae I and II) and around the central canal (lamina X). COX‐2‐li was also observed in some neurones in deep dorsal horn and in individual motor neurones in ventral horn. COX‐2‐li was not observed in the cell bodies of DRG. 6 Superfusion of the lumbar spinal cord of normal rats with artificial CSF and subsequent radioimmunoassay revealed the presence of prostaglandin D2 (PGD2)

Characterization of cultured dorsal root ganglion neuron P2X receptors

P2X receptors for adenosine 5′‐triphosphate (ATP) comprise a family of ligand‐gated cation channels with distinct characteristics which are dependent on the receptor subunits (P2X1–7) expressed, and the homomeric or heteromeric assembly of protein subunits in individual cells. We describe the properties of P2X receptors expressed by cultured adult rat dorsal root ganglion cells on the basis of the time course of responses to ATP, α,β‐methylene adenosine 5′‐triphosphate (α,β‐meATP) and 2‐methyl‐thioadenosine 5′‐triphosphate (2‐meSATP), and using the antagonists 2′,3′‐O‐(2,4,6‐trinitrophenyl) ATP (TNP‐ATP), a novel and highly selective purinoceptor antagonist, suramin and iso‐pyridocalphosphate‐6‐azophenyl‐2′,5′ disulphonic acid (PPADS). ATP (10 μm) evoked inward currents in ≈ 95% of neurons tested and > 80% responded with a fast transient inward current that rapidly inactivated during the continued presence of ATP. Of the remaining neurons, ≈ 4% showed a sustained response and ≈ 10% showed a combination of transient and sustained components. Rapid application of ATP, α,β‐meATP and 2meSATP demonstrated these to be full agonists of the rapidly inactivating P2X receptor (pA50 values = 5.83, 5.86 and 5.55, respectively), whilst uridine triphosphate (UTP) and 1‐β,γ‐methyleneadenosine 5′‐triphosphate (1‐β,γ‐meATP) were ineffective as agonists. These rapidly inactivating responses could be inhibited by TNP‐ATP, suramin and PPADS (pIC50 = 9.5, 6.5, 6.4, respectively). Using inactivation protocols, we demonstrate the presence of homomeric P2X3‐like receptors and non‐inactivating P2X receptors, which indicates that individual subsets of adult dorsal root ganglion neurons have distinct P2X receptor phenotypes, and that individual DRG neurons may express multiple P2X receptor subtypes.

The intraspinal release of prostaglandin E2 in a model of acute arthritis is accompanied by an up-regulation of cyclo-oxygenase-2 in the spinal cord

In anaesthetized rats, the intraspinal release of immunoreactive prostaglandin E2 was measured using antibody microprobes. We addressed the question of whether the release of immunoreactive prostaglandin E2 is altered during development of acute inflammation in the knee evoked by intra-articular injections of kaolin and carrageenan. We also examined cyclo-oxygenase-1 and cyclo-oxygenase-2 protein levels in the spinal cord during the development of inflammation using the same model of arthritis. Densitometric analysis of microprobes showed that basal release of immunoreactive prostaglandin E2 in the period 175-310 min after kaolin was slightly higher than in the absence of inflammation. A pronounced enhancement of basal release of immunoreactive prostaglandin E2 was observed 430-530 min after kaolin. Enhanced levels of immunoreactive prostaglandin E2 were observed throughout the dorsal and ventral horns. Release of immunoreactive prostaglandin E2 was not altered further by the application of innocuous and noxious pressure onto the inflamed knee. Western blot analysis revealed that cyclo-oxygenase-2 but not cyclo-oxygenase-1 protein levels were elevated in the spinal cords of animals with inflammation compared to normal animals. This effect was evident as early as 3 h after the induction of arthritis. The maximum elevation of cyclo-oxygenase-2 protein levels (six-fold) was observed 12 h after the induction of arthritis. The results show that there is a tonic release of immunoreactive prostaglandin E2 from the spinal cord following the induction of arthritis, which is accompanied by enhanced expression of cyclo-oxygenase-2 protein in the spinal cord. We suggest that intraspinal prostaglandins may play a role in inflammation-evoked central sensitization of spinal cord neurons.

The Effects of NMDA Antagonists on Neuronal Activity in Cat Spinal Cord Evoked by Acute Inflammation in the Knee Joint

In α‐chloralose‐anaesthetized, spinalized cats we examined the effects of NMDA antagonists on the discharges of 71 spinal neurons which had afferent input from the knee joint. These neurons were rendered hyperexcitable by acute arthritis in the knee induced by kaolin and carrageenan. They were located in the deep dorsal and ventral horn and some of them had ascending axons. The N‐methyl‐d‐aspartate (NMDA) antagonists ketamine and d‐2‐amino‐5‐phosphonovalerate (AP5), were administered ionophoretically, and ketamine was also administered intravenously. In some of the experiments the antagonists were tested against the agonists NMDA and quisqualate. The effects of the NMDA antagonists consisted of a significant reduction in the resting activity of neurons and/or the responses of the same neurons to mechanical stimulation of the inflamed knee. Intravenous ketamine was most effective in suppressing the resting and mechanically evoked activity in 25 of 26 neurons tested. Ionophoretically applied ketamine had a suppressive effect in 11 of 21 neurons, and AP5 decreased activity in 17 of 24 cells. The reduction in the resting and/or the mechanically evoked discharges was achieved with doses of the antagonists which suppressed the responses to NMDA but not those to quisqualate. These results suggest that NMDA receptors are involved in the enhanced responses and basal activity of spinal neurons induced by inflammation in the periphery.

Acute and chronic phases of unilateral inflammation in rat's ankle are associated with an increase in the proportion of calcitonin gene-related peptide-immunoreactive dorsal root ganglion cells

Using immunocytochemical methods, the proportion of calcitonin gene‐related peptide immunoreactive perikarya was determined in dorsal root ganglia L4–L6 in four control rats and in ten rats with a unilateral inflammation in the ankle region of the left hindlimb. The inflammation was induced by subdermal injection of Freunds complete adjuvant at the ankle. Swelling and cellular infiltration of the ankle region developed within 2 days, and were stable and restricted to the injected ankle for the duration of the 3‐week study. In control rats ∼24% of 20 419 perikarya showed calcitonin gene‐related peptide (CGRP)‐like immunoreactivity. In rats with unilateral inflammation the proportion of CGRP‐positive neurons was increased on the inflamed side to ∼32% of 11 454 cells at day 2 (P < 0.001 with respect to ganglia in normal rats) and ∼29% of 10 739 perikarya at day 20 post inoculation (P < 0.01). By contrast, no significant changes were found between ganglia in the non‐injected side (∼25% at day 2 and ∼24% at day 20). These results demonstrate that peripheral inflammation is associated with an increase in the proportion of neurons in the dorsal root ganglia that synthetize CGRP. This up‐regulation is already present at an early stage of inflammation but also at later stages, suggesting that the increased synthesis of CGRP is an important neurobiological reaction associated with the acute and chronic phases of inflammation.

Cyclooxygenase‐1 is a marker for a subpopulation of putative nociceptive neurons in rat dorsal root ganglia

Immunocytochemical and morphometric techniques were used to quantify the distribution of cyclooxygenase (cox)‐containing neurons in rat L5 dorsal root ganglia (DRG). Cox‐1 immunolabelling was almost exclusively restricted to small diameter DRG neurons (< 1000 μm2), and was extensively colocalized with calcitonin gene‐related peptide (CGRP) and isolectin B4 (IB4). Cox‐1 was present in 65% and 70% of CGRP‐ and IB4‐labelled neurons, respectively. Cox‐1 labelling was also found in neurons expressing the sensory neuron‐specific (SNS) Na+ channel. Cox‐2 labelling was absent in DRG from normal rats. In the Freunds adjuvant model of monoarthritis, the proportion of cox‐1‐positive DRG neurons was unchanged and no neurons were found to be labelled for cox‐2. In primary tissue culture, cox‐1 immunolabelling persisted in vitro for up to 9 days and was present in morphologically identical neurons. The selective expression of cox‐1 in peripheral ganglia was confirmed by the small number of nodose ganglion neurons and superior cervical ganglion (SCG) neurons labelled for cox‐1. These data suggest that cox‐1 is a marker for a subpopulation of putative nociceptive neurons in vitro and in vivo, and suggests that the prostaglandins synthesized by these neurons may be important for nociceptor function. These data may have important implications for the mode and mechanism of action of non‐steroidal anti‐inflammatory drugs (NSAIDs).

The distribution of small and intermediate conductance calcium-activated potassium channels in the rat sensory nervous system

Small (SK) and intermediate (IK) conductance calcium-activated potassium channels are candidate ion channels for the regulation of excitability in nociceptive neurones. We have used unique peptide-directed antisera to describe the immunocytochemical distribution of the known isoforms of these ion channels in dorsal root ganglia (DRG) and spinal cord of the rat. These investigations sought to characterize further the phenotype and hence possible functions of nociceptive neurone subpopulations in the rat. In addition, using Western blotting, we sought to determine the level of protein expression of SK and IK channels in sensory nervous tissues following induction of inflammation (Freunds Complete Adjuvant (FCA) arthritis model) or nerve injury (chronic constriction injury model). We show that SK1, SK2, SK3 and IK1 are all expressed in DRG and spinal cord. Morphometric analysis revealed that SK1, SK2 and IK1 were preferentially localized to neurones having cell bodies <1000 microm2 (putative nociceptors) in DRG. Dual labeling immunocytochemistry showed that these ion channels co-localize with both CGRP and IB4, known markers of nociceptor sub-populations. SK2 was localized almost exclusively in the superficial laminae of the spinal cord dorsal horn, the region in which many sensory afferents terminate; the distribution of SK1 and IK1 was more widespread in spinal cord, although some preferential labeling within the dorsal horn was observed in the case of IK1. Here we show evidence for a distinctive pattern of expression for certain members of the calcium-activated potassium channel family in the rat DRG.

Cyclooxygenase-1 is a marker for a subpopulation of putative nociceptive neurons in rat dorsal root ganglia

Immunocytochemical and morphometric techniques were used to quantify the distribution of cyclooxygenase (cox)‐containing neurons in rat L5 dorsal root ganglia (DRG). Cox‐1 immunolabelling was almost exclusively restricted to small diameter DRG neurons (< 1000 μm2), and was extensively colocalized with calcitonin gene‐related peptide (CGRP) and isolectin B4 (IB4). Cox‐1 was present in 65% and 70% of CGRP‐ and IB4‐labelled neurons, respectively. Cox‐1 labelling was also found in neurons expressing the sensory neuron‐specific (SNS) Na+ channel. Cox‐2 labelling was absent in DRG from normal rats. In the Freunds adjuvant model of monoarthritis, the proportion of cox‐1‐positive DRG neurons was unchanged and no neurons were found to be labelled for cox‐2. In primary tissue culture, cox‐1 immunolabelling persisted in vitro for up to 9 days and was present in morphologically identical neurons. The selective expression of cox‐1 in peripheral ganglia was confirmed by the small number of nodose ganglion neurons and superior cervical ganglion (SCG) neurons labelled for cox‐1. These data suggest that cox‐1 is a marker for a subpopulation of putative nociceptive neurons in vitro and in vivo, and suggests that the prostaglandins synthesized by these neurons may be important for nociceptor function. These data may have important implications for the mode and mechanism of action of non‐steroidal anti‐inflammatory drugs (NSAIDs).

The involvement of N-methyl-d-aspartate (NMDA) and non-NMDA receptors in the responsiveness of rat spinal neurons with input from the chronically inflamed ankle

Unilateral adjuvant inflammation was induced at the rat ankle 2 or 20 days before an evaluation of the contribution of N-methyl-D-aspartate (NMDA) and non-NMDA receptors to the processing of nociceptive information by wide dynamic range neurons in the spinal cord. Microionophoretic application of either the NMDA receptor antagonists ketamine and DL-2-amino-5-phosphonovalerate (AP5) or the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) reduced the responses to innocuous and noxious mechanical stimulation of the inflamed ankle. The pattern of these effects was comparable to that in rats with acute inflammation suggesting that non-NMDA and NMDA receptors are similarly involved in acute, prolonged acute and chronic inflammation-evoked activity.

Expression and regulation of prostaglandin E receptor subtype mRNAs in rat sensory ganglia and spinal cord in response to peripheral inflammation

Prostaglandins are known to act via seven transmembrane domain receptors to exert actions on both peripheral and central neurons resulting in changes in neuronal excitability. Prostaglandin E2, the prostaglandin most often associated with inflammation, itself acts on a family of closely related receptors, the EP receptors. Using semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), we have shown that rat primary afferent neurons express the mRNA for all EP receptor subtypes, and that some, but not all EP receptor subtype mRNAs are down-regulated in sensory neurons in response to an acute peripheral inflammation. We also show for the first time that all EP receptor subtype mRNAs are expressed in rat lumbar spinal cord. Spinal cord EP receptor subtype mRNAs are also regulated in acute inflammation in a pattern distinct from the changes seen in sensory ganglia in response to the same inflammatory stimulus.