mRNA In Spinal Cord Research Articles

γ-Aminobutyric Acid Transporter 1 Negatively Regulates T Cell-Mediated Immune Responses and Ameliorates Autoimmune Inflammation in the CNS

gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter of the CNS, and GABA transporter 1 (GAT-1) is critical in maintaining a GABA reservoir and associated functions. The wide expression of GAT-1 in the CNS prompted us to explore its role in neuroimmunological disorders. In mice induced with experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis, we found that the expression levels of GAT-1 mRNA and protein in spinal cord were greatly suppressed as compared with those in naive mice and irrelevant Ag-immunized mice. Therefore, we induced EAE in GAT-1(-/-) mice and found that the disease was significantly aggravated and was accompanied by some nonclassic EAE signs. Mononuclear cells from GAT-1(-/-) mice with EAE showed much higher Ag-specific proliferative responses. Proinflammatory cytokine production in these mice was also greatly up-regulated. Further studies revealed that GAT-1 deficiency induced vigorous immune responses by enhancing IkappaB kinase phosphorylation and NF-kappaB-DNA binding activity, as well as strengthening the T-bet-STAT1 circuit signaling pathway. Finally, we found that GAT-1 was expressed only on activated T cells primed with Ags, but not on B cells or macrophages. These findings indicate that GAT-1 is a critical modulator in T cell-mediated immune responses and in EAE pathogenesis.

Spinal Cord mRNA Profile in Patients with ALS: Comparison with Transgenic Mice Expressing the Human SOD-1 Mutant

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by loss of motor neurons in the cerebral cortex, brain stem, and spinal cord. Most cases (90%) are classified as sporadic ALS (sALS). The remainder 10% are inherited and referred to as familial ALS, and 2% of instances are due to mutations in Cu/Zn superoxide dismutase (SOD1). Using cDNA microarray on postmortem spinal cord specimens of four sALS patients compared to four age-matched nonneurological controls, we found major changes in the expression of mRNA in 60 genes including increase of cathepsin B and cathepsin D (by the factors 2 and 2.3, respectively), apolipoprotein E (Apo E; factor 4.2), epidermal growth factor receptor (factor 10), ferritin (factor 2), and lysosomal trafficking regulator (factor 10). The increase in the expression of these genes was verified by quantitative reverse transcriptase polymerase chain reaction. Further analysis of these genes in hSOD1-G93A transgenic mice revealed increase in the expression in parallel with the deterioration of motor functions quantified by means of rotorod performance. The comparability of the findings in sALS patients and in the hSOD1-G93A transgenic mouse model suggests that the examined genes may play a specific role in the pathogenesis of ALS.

Blockade of sensory abnormalities and kinin B1 receptor expression by N-Acetyl-l-Cysteine and ramipril in a rat model of insulin resistance

Glucose-fed rat is a model of insulin resistance that displays sensory polyneuropathy and hypertension. This study aimed at comparing the beneficial effects of N-Acetyl-l-Cysteine (NAC, antioxidant) and ramipril (angiotensin-1 converting enzyme inhibitor) on tactile and cold allodynia induced by chronic glucose feeding. Impact of these treatments was also assessed on hypertension, plasma glucose and insulin concentrations, insulin resistance and kinin B1 receptor expression.Male Wistar rats (50–75 g) were given 10% d-glucose in their drinking water for 11 weeks or tap water (controls). Glucose-fed rats were treated either with NAC (1 g/kg/day, gavage), ramipril (1 mg/kg/day in drinking water) or no drug during the last 5 weeks. Glucose feeding for 6 weeks induced a significant increase in systolic blood pressure and hyperglycaemia which was accompanied by tactile and cold allodynia. At 11 weeks, plasma insulin, insulin resistance (HOMA index), kinin B1 receptor mRNA in spinal cord and renal cortex and B1 receptor binding sites in spinal cord were enhanced in glucose-fed rats. NAC and ramipril caused a progressive to complete inhibition of tactile and cold allodynia from 6 to 11 weeks. High systolic blood pressure, hyperinsulinemia, insulin resistance and kinin B1 receptor expression were also normalized or attenuated in glucose-fed rats by either treatment. Results suggest that chronic treatment with an antioxidant or an ACE inhibitor provides similar beneficial effects on sensory polyneuropathy, hypertension and insulin resistance in glucose-fed rats. Both therapies were associated with a reduction of the expression of the pro-nociceptive kinin B1 receptor.

Neurobin/TMPRSS11c, a novel type II transmembrane serine protease that cleaves fibroblast growth factor-2 in vitro

TTSPs [type II TMPRSSs (transmembrane serine proteases)] are a growing family of trypsin-like enzymes with, in some cases, restricted tissue distribution. To investigate the expression of TTSPs in the nervous system, we performed a PCR-based screening approach with P10 (postnatal day 10) mouse spinal cord mRNA. We detected the expression of five known TTSPs and identified a novel TTSP, which we designated neurobin. Neurobin consists of 431 amino acids. In the extracellular part, neurobin contains a single SEA (sea-urchin sperm protein, enterokinase and agrin) domain and a C-terminal serine protease domain. RT-PCR (reverse transcription-PCR) analysis indicated the expression of neurobin in spinal cord and cerebellum. Histochemical analysis of brain sections revealed distinct staining of Purkinje neurons of the cerebellum. Transiently overexpressed neurobin was autocatalytically processed and inserted into the plasma membrane. Autocatalytic activation could be suppressed by mutating Ser(381) in the catalytic pocket to an alanine residue. The protease domain of neurobin, produced in Escherichia coli and refolded from inclusion bodies, cleaved chromogenic peptides with an arginine residue in position P(1). Serine protease inhibitors effectively suppressed the proteolytic activity of recombinant neurobin. Ca2+ or Na+ ions did not significantly modulate the catalytic activity of the protease. Recombinant neurobin processed 17-kDa FGF-2 (fibroblast growth factor-2) at several P(1) lysine and arginine positions to distinct fragments, in a heparin-inhibitable manner, but did not cleave FGF-7, laminin or fibronectin. These results indicate that neurobin is an authentic TTSP with trypsin-like activity and is able to process FGF-2 in vitro.

Transmembrane protein 50b (C21orf4), a candidate for Down syndrome neurophenotypes, encodes an intracellular membrane protein expressed in the rodent brain

Transmembrane protein 50b, Tmem50b, previously referred to as C21orf4, encodes a predicted transmembrane protein and is one of few genes significantly over-expressed during cerebellar development in a Down syndrome mouse model, Ts1Cje. In order to assess potential mechanisms by which Tmem50b could contribute to Down syndrome–related phenotypes, we determined the expression patterns of Tmem50b mRNA, as well as Tmem50b protein distribution, expression and subcellular localization. In situ hybridization in mice at embryonic day 14.5 showed cortical plate and spinal cord mRNA expression. By postnatal day 7, strong mRNA expression was seen in the cerebellum, hippocampus and olfactory bulb, with diffuse cortical expression. Quantitative PCR of adult mouse tissue showed Tmem50b mRNA expression in the brain, heart and testis. A rabbit polyclonal antibody was generated against Tmem50b and rat and mouse tissue screening by Western blot, and immunohistochemistry showed that protein expression concurred with mRNA expression. Double immunofluorescence revealed that Tmem50b is highly expressed in rat and mouse glial fibrillary acidic protein–positive cells in vivo and in vitro, but less so in neuronal MAP2- or β-tubulin II-positive cells in vitro. Tmem50b is invariably expressed in cultured mouse neural precursor cells. In adult mouse cerebellum sections, Tmem50b immunoreactivity was found in Purkinje and Golgi cell somata and in Bergmann glial processes. Electron microscopy confirmed that Tmem50b was present on endoplasmic reticulum (ER) and Golgi apparatus membranes. Results indicate that Tmem50b is a developmentally-regulated intracellular ER and Golgi apparatus membrane protein that may prove important for correct brain development through functions associated with precursor cells and glia.

The effect of 4-Methylcatechol treatment in chronic constrictive injury of the rat sciatic nerve on the allodynia and spinal neurotrophic factors

Background: The effect of chronic administration of 4-methylcatechol known as a neurotrophic factor inducer on the allodynia and spinal neurotrophic factors was investigated in chronic constrictive injury of sciatic nerve in rats. Methods: With the Sprague Dawly rat, sciatic nerve was loosely ligated with 4-0 chromic catgut and neuropathic pain model was made. The threshold for tactile allodynia was measured with von Frey hair by up-down method and cold allodynia was measured by dropping 20μl of 100% acetone on the dorsum of the injured foot. 4-Methylcatechol (100μg/kg, intraperitoneal) was injected once a day for 14 days and the effect on allodynia was compared with saline injected group. At 3, 7 and 14 days after injection, lumbar spinal cord was harvested and the mRNA content of nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) was measure by real time PCR. Results: Mechanical and cold allodynia improved from 7 days after 4-methylcatechol administration. NGF and BDNF in spinal cord decreased compared to sham operated group. BDNF in lumbar spinal cord has increased tendency after treatment without statistical significance. Conclusions: Chronic intraperitoneal administration of 4-methylcatechol may improve tactile and cold allodynia in chronic constrictive injury rat model of neuropathic pain. The BDNF mRNA in spinal cord might increase after 4-methylcatechol treatment.

Single-Channel Recording of TASK-3-like K+ Channel and Up-Regulation of TASK-3 mRNA Expression after Spinal Cord Injury in Rat Dorsal Root Ganglion Neurons

Single-channel recordings of TASK-1 and TASK-3, members of two-pore domain K(+) channel family, have not yet been reported in dorsal root ganglion (DRG) neurons, even though their mRNA and activity in whole-cell currents have been detected in these neurons. Here, we report single-channel kinetics of the TASK-3-like K(+) channel in DRG neurons and up-regulation of TASK-3 mRNA expression in tissues isolated from animals with spinal cord injury (SCI). In DRG neurons, the single-channel conductance of TASK-3-like K(+) channel was 33.0+/-0.1 pS at -60 mV, and TASK-3 activity fell by 65+/-5% when the extracellular pH was changed from 7.3 to 6.3, indicating that the DRG K(+) channel is similar to cloned TASK-3 channel. TASK-3 mRNA and protein levels in brain, spinal cord, and DRG were significantly higher in injured animals than in sham-operated ones. These results indicate that TASK-3 channels are expressed and functional in DRG neurons and the expression level is up-regulated following SCI, and suggest that TASK-3 channel could act as a potential background K(+) channel under SCI-induced acidic condition.

Interleukin-6 mediates low-threshold mechanical allodynia induced by intrathecal HIV-1 envelope glycoprotein gp120

Spinal cord glia (microglia and astrocytes) contribute to enhanced pain states. One model that has been used to study this phenomenon is intrathecal (i.t.) administration of gp120, an envelope glycoprotein of HIV-1 known to activate spinal cord glia and thereby induce low-threshold mechanical allodynia, a pain symptom where normally innocuous (non-painful) stimuli are perceived as painful. Previous studies have shown that i.t. gp120-induced allodynia is mediated via the release of the glial pro-inflammatory cytokines, tumor necrosis factor-α (TNF), and interleukin-1β (IL-1). As we have recently reported that i.t. gp120 induces the release of interleukin-6 (IL-6), in addition to IL-1 and TNF, the present study tested whether this IL-6 release in spinal cord contributes to gp120-induced mechanical allodynia and/or to gp120-induced increases in TNF and IL-1. An i.t. anti-rat IL-6 neutralizing antibody was used to block IL-6 actions upon its release by i.t. gp120. This IL-6 blockade abolished gp120-induced mechanical allodynia. While the literature predominantly documents the cascade of pro-inflammatory cytokines as beginning with TNF, followed by the stimulation of IL-1, and finally TNF plus IL-1 stimulating the release of IL-6, the present findings indicate that a blockade of IL-6 inhibits the gp120-induced elevations of TNF, IL-1, and IL-6 mRNA in dorsal spinal cord, elevation of IL-1 protein in lumbar dorsal spinal cord, and TNF and IL-1 protein release into the surrounding lumbosacral cerebrospinal fluid. These results would suggest that IL-6 induces pain facilitation, and may do so in part by stimulating the production and release of other pro-inflammatory cytokines.

Distribution of the iron‐regulating protein hepcidin in the murine central nervous system

Iron serves as an essential trace element for all body tissues, including the central nervous system (CNS). Because iron deficiency as well as iron overload is known to cause damage to the mammalian brain, the maintenance of iron homeostasis is crucial. It has been discovered recently that hepcidin plays an essential role in iron metabolism outside the CNS. A defect in hepcidin expression is responsible for iron accumulation and mice over-expressing hepcidin die postnatally by a severe anemia. We have used RT-PCR, in situ hybridization, and immunohistochemistry to investigate the cellular distribution of hepcidin mRNA and protein in brain, spinal cord, and dorsal root ganglia. Our results show a wide-spread distribution of hepcidin in different brain areas, including the olfactory bulb, cortex, hippocampus, amygdala, thalamus, hypothalamus, mesencephalon, cerebellum, pons, spinal cord, as well as in dorsal root ganglia of the peripheral nervous system. Hepcidin immunoreactivity is not restricted to neurons, but can be detected in both neurons and GFAP-positive glia cells. Because hepcidin action in organs outside the CNS is linked to iron homeostasis, we speculate that it is also involved in such processes in the CNS, putatively together with other iron regulating proteins. Cellular mechanisms and functions of hepcidin in the CNS remain to be elucidated.

Systemic inflammation induces COX-2 mediated prostaglandin D2 biosynthesis in mice spinal cord

Although prostaglandin (PG)D 2 is one of the main metabolites of the cyclooxygenase (COX) pathway of arachidonate metabolism in the brain, relatively little is known about the regulation of PGD 2 biosynthesis in the spinal cord during systemic inflammation. Therefore, the present study was aimed at investigating the effect of endotoxin treatment on spinal PGD 2 biosynthesis in BALB/c mice. Spinal inflammatory response to systemic endotoxin was verified by determination of spinal TNFα and IL-1β mRNA. COX-1, COX-2, membrane-bound prostaglandin E synthase-1 (mPGES-1), and lipocalin-type prostaglandin D synthase (L-PGDS) mRNA and protein were determined by RT-PCR and western blot, respectively. The concentrations of immunoreactive PGD 2 and PGE 2 were measured in superfusion media of spinal cord samples in-vitro. Endotoxin treatment (1 mg/kg; 24 h before) enhanced the expression of COX-2, mPGES-1, and L-PGDS mRNA and protein in spinal cord, while there was no significant effect on COX-1 mRNA and protein. In superfusion media of spinal cord samples obtained from endotoxin treated mice, the concentrations of immunoreactive PGE 2 and PGD 2 were higher than in the control group suggesting enhanced spinal PG biosynthesis after endotoxin treatment. Addition of the selective COX-2 inhibitor lumiracoxib (100 nM) to the superfusion medium did not significantly affect PGE 2 or PGD 2 release in spinal cord obtained from non-treated mice. In spinal cord of endotoxin-treated mice, lumiracoxib (100 nM) attenuated PGE 2 and PGD 2 release to values similar to those observed in tissue obtained from non-endotoxin-treated mice. These results show enhanced expression of spinal L-PGDS and increased spinal PGD 2 biosynthesis during systemic inflammation whereby enhanced biosynthesis seems to be dependent primarily on COX-2 activity.

Reduced Nerve Injury-Induced Neuropathic Pain in Kinin B1Receptor Knock-Out Mice

Injury to peripheral nerves often results in a persistent neuropathic pain condition that is characterized by spontaneous pain, allodynia, and hyperalgesia. Nerve injury is accompanied by a local inflammatory reaction in which nerve-associated and immune cells release several pronociceptive mediators. Kinin B1 receptors are rarely expressed in nontraumatized tissues, but they can be expressed after tissue injury. Because B1 receptors mediate chronic inflammatory painful processes, we studied their participation in neuropathic pain using receptor gene-deleted mice. In the absence of neuropathy, we found no difference in the paw-withdrawal responses to thermal or mechanical stimulation between B1 receptor knock-out mice and 129/J wild-type mice. Partial ligation of the sciatic nerve in the wild-type mouse produced a profound and long-lasting decrease in thermal and mechanical thresholds in the paw ipsilateral to nerve lesion. Threshold changed neither in the sham-operated animals nor in the paw contralateral to lesion. Ablation of the gene for the B1 receptor resulted in a significant reduction in early stages of mechanical allodynia and thermal hyperalgesia. Furthermore, systemic treatment with the B1 selective receptor antagonist des-Arg9-[Leu8]-bradykinin reduced the established mechanical allodynia observed 7-28 d after nerve lesion in wild-type mice. Partial sciatic nerve ligation induced an upregulation in B1 receptor mRNA in ipsilateral paw, sciatic nerve, and spinal cord of wild-type mice. Together, kinin B1 receptor activation seems to be essential to neuropathic pain development, suggesting that an oral-selective B1 receptor antagonist might have therapeutic potential in the management of chronic pain.

Identification and Functional Characterization of a 5-Transmembrane Domain Variant Isoform of the NTS2 Neurotensin Receptor in Rat Central Nervous System

The present study demonstrated that alternative splicing of the rat nts2 receptor gene generates a 5-transmembrane domain variant isoform (vNTS2) that is co-expressed with the full-length NTS2 receptor throughout the brain and spinal cord, as evidenced by reverse transcription-PCR. The vNTS2 polypeptide is 281 amino acids in length, which is 135 amino acids shorter than the full-length isoform. Immunohistochemical and radioligand binding studies revealed that the HA-tagged recombinant vNTS2 receptor is poorly targeted to plasma membranes in transfected COS-7 cells. Binding studies also showed that the truncated receptor displayed a 5000-fold lower affinity for neurotensin (NT) than its full-length counterpart (IC(50) of 10 mum and 2 nm, respectively). Yet NT binding induced efficient internalization of receptor-ligand complexes in vNTS2-transfected cells. Furthermore, it produced a rapid (<5 min) activation of the mitogen-activated protein kinases (ERK1/2) pathway, indicating functional coupling of the variant receptor. This activation is sustained (>1 h) and is also produced by the NTS2 agonist levocabastine. Western blotting experiments suggested that vNTS2 is not expressed in monomeric form in the rat central nervous system. However, it does appear to form a variety of multimeric complexes, including homodimers and heterodimers, with the full-length NTS2. Indeed, co-immunoprecipitation studies in dually transfected cells demonstrated that the two receptor isoforms can form stable associations. Taken together, the present results indicated that the rat vNTS2 is a functional receptor that may play a role in NT signaling in mammalian central nervous system.

Androgenic down-regulation of urotensin II precursor, urotensin II-related peptide precursor and androgen receptor mRNA in the mouse spinal cord

We had previously shown that in rat spinal cord motoneurons urotensin II (UII) precursor mRNA was down-regulated by androgens. Very recently, a gene encoding the precursor of a UII analog, termed UII-related peptide (URP), has been identified. Using in situ hybridization, we studied the localization of UII and URP precursor as well as androgen receptor (AR) mRNA in the male mouse thoracic spinal cord. We also evaluated the androgenic regulation of the two peptide precursor and AR mRNA expression in the ventral horn of the mouse thoracic spinal cord. The results revealed that URP precursor mRNA was localized in motoneurons and that the vast majority of the motoneurons expressed both peptide precursor as well as AR mRNA. Seven-day castration induced an increase in UII and URP precursor and AR mRNA levels. Short term (3–24 h) administration of dihydrotestosterone to castrated animals restored the three protein mRNA levels to the levels observed in intact animals. These results suggest that in the ventral horn of the mouse spinal cord both UII and URP precursor and AR mRNA are expressed by the same neurons and that circulating androgens are exerting a down-regulation of the three protein mRNA expression, possibly by a direct action on motoneurons.

Spinal distribution and metabolism of 2′- O-(2-methoxyethyl)-modified oligonucleotides after intrathecal administration in rats

Intrathecal (IT) delivery of antisense oligodeoxynucleotides (ASO) has been used to study the function of specific gene products in spinal nociception. However, a lack of systematic studies on the spinal distribution and kinetics of IT ASO is a major hurdle to the utilization of this technique. In the present study, we injected rats IT with 2′- O-(2-methoxyethyl) modified phosphorothioate ASO (2′- O-MOE ASO) and examined anatomical and cellular location of the ASO in the spinal cord and dorsal root ganglia (DRG) by immunocytochemistry. At 0.5 h after a single IT injection, immunostaining for ISIS 13920 (a 2′- O-MOE ASO targeting h-ras) localized superficially in the lumbar spinal cord, while at 24 h the immunostaining was distributed throughout the spinal cord and was predominantly intracellular. Double staining with cell type specific antibodies indicated that the ASO was taken up by both glia and neurons. ASO immunoreactivity was also observed in DRG after IT ISIS 13920. Capillary gel electrophoresis analysis showed that ISIS 22703, a 2′- O-MOE ASO targeting the α isozyme of protein kinase C (PKC), remained intact in spinal cord tissue and cerebrospinal fluid up to 24 h after the injection and no metabolites were detected. In contrast, after IT ISIS 11300, an unmodified phosphorothioate ASO with the same sequence as ISIS 22703, no full-length compound was detectable at 24 h, and metabolites were seen as early as 0.5 h. IT treatment with ISIS 22703 at doses that effectively down-regulated PKCα mRNA in spinal cord did not affect the mRNA expression in DRG. In summary, 2′- O-MOE ASO displayed high stability in spinal tissue after IT delivery, efficiently distributed to spinal cord, and internalized into both neuronal and non-neuronal cells. ASO are able to reach DRG after IT delivery; however, higher doses may be required to reduce target gene in DRG as compared with spinal cord.

Formalin Injection Causes a Coordinated Spinal Cord CO/NO-cGMP Signaling System Response

BackgroundThe CO/NO-cGMP signalling system participates in the regulation of many physiological processes. The roles this system plays in spinal cord nociceptive signalling are particularly important. While individual components have been examined in isolation, little study has been dedicated to understanding the regulation and functioning of the system as a whole.ResultsIn these studies we examined the time course of expression of 13 genes coding for components of this system including isoforms of the heme oxygenase (HO), nitric oxide synthase (NOS), soluble guanylate cyclase (sGC), cGMP dependent protein kinase (PKG) and phosphodiesterase (PDE) enzyme systems. Of the 13 genes studied, 11 had spinal cord mRNA levels elevated at one or more time points up to 48 hours after hindpaw formalin injection. Of the 11 with elevated mRNA, 8 had elevated protein levels 48 hours after formalin injection when mechanical allodynia was maximal. No component had an increased protein level which did not have an increased mRNA level at one or more time points. Injection of morphine 10 mg/kg prior to formalin completely abolished the acute nociceptive behaviours, but did not alter the degree of sensitivity which developed in the formalin treated hind paws during the subsequent 48 hours. Morphine treatment did, however, eliminate formalin induced increases in enzyme protein levels.ConclusionOur results indicate that the expression of the components of the CO/NO-cGMP signalling system seems to be coordinated in such a way that a generalized multi-level enhancement rather than a tightly limited step specific response occurs with noxious stimulation. Furthermore, the analgesic morphine administered prior to noxious stimulation can prevent long-term changes in gene expression though not necessarily nociceptive sensitisation.

A role for proinflammatory cytokines and fractalkine in analgesia, tolerance, and subsequent pain facilitation induced by chronic intrathecal morphine.

The present experiments examined the role of spinal proinflammatory cytokines [interleukin-1beta (IL-1)] and chemokines (fractalkine) in acute analgesia and in the development of analgesic tolerance, thermal hyperalgesia, and tactile allodynia in response to chronic intrathecal morphine. Chronic (5 d), but not acute (1 d), intrathecal morphine was associated with a rapid increase in proinflammatory cytokine protein and/or mRNA in dorsal spinal cord and lumbosacral CSF. To determine whether IL-1 release modulates the effects of morphine, intrathecal morphine was coadministered with intrathecal IL-1 receptor antagonist (IL-1ra). This regimen potentiated acute morphine analgesia and inhibited the development of hyperalgesia, allodynia, and analgesic tolerance. Similarly, intrathecal IL-1ra administered after the establishment of morphine tolerance reversed hyperalgesia and prevented the additional development of tolerance and allodynia. Fractalkine also appears to modulate the effects of intrathecal morphine because coadministration of morphine with intrathecal neutralizing antibody against the fractalkine receptor (CX3CR1) potentiated acute morphine analgesia and attenuated the development of tolerance, hyperalgesia, and allodynia. Fractalkine may be exerting these effects via IL-1 because fractalkine (CX3CL1) induced the release of IL-1 from acutely isolated dorsal spinal cord in vitro. Finally, gene therapy with an adenoviral vector encoding for the release of the anti-inflammatory cytokine IL-10 also potentiated acute morphine analgesia and attenuated the development of tolerance, hyperalgesia, and allodynia. Taken together, these results suggest that IL-1 and fractalkine are endogenous regulators of morphine analgesia and are involved in the increases in pain sensitivity that occur after chronic opiates.

PACAP mRNA is expressed in rat spinal cord neurons

This study examines the expression of pituitary adenylate cyclase activating polypeptide (PACAP) mRNA in the rat spinal cord during normal conditions and in response to sciatic nerve transection. Previously, PACAP immunoreactivity has been found in fibers in the spinal cord dorsal horn and around the central canal and in neurons in the intermediolateral column (IML). Furthermore, in the dorsal root ganglia, PACAP immunoreactivity and PACAP mRNA expression have been observed preferentially in nerve cell bodies of smaller diameter terminating in the superficial laminae of the dorsal horn. However, neuronal expression of PACAP mRNA in adult rat spinal cord appeared limited to neurons of the IML. By using a refined in situ hybridization protocol, we now detect PACAP mRNA expression in neurons primarily in laminae I and II, but also in deeper laminae of the spinal cord dorsal horn and around the central canal. In addition, PACAP mRNA expression is observed in a few neurons in the ventral horn. PACAP expression in the ventral horn is increased in a population of large neurons, most likely motor neurons, both after distal and proximal sciatic nerve transection. The proposed role of PACAP in nociception is strengthened by our findings of PACAP mRNA-expressing neurons in the superficial laminae of the dorsal horn. Furthermore, increased expression of PACAP in ventral horn neurons, in response to nerve transection, suggests a role for PACAP in repair/regeneration of motor neurons.

Up-regulation of metabotropic glutamate receptor subtypes 3 and 5 in spinal cord in a clinical model of persistent inflammation and hyperalgesia

Evidence from experimental pain research has revealed that metabotropic glutamate receptors (mGluRs) play a pivotal role in nociceptive processing, inflammatory pain and hyperalgesia. The aim of this study was to characterise expression of group I and II mGluRs in spinal cord in a model of naturally occurring persistent inflammation (sheep with unilateral lameness due to inflammation of the digital tissues of the feet, estimated to have been affected by the condition for >2 weeks) and an experimental model of acute inflammation (injection of intradermal carrageenan into lower forelimb in sheep). Animals with unilateral clinical inflammation displayed significant mechanical hyperalgesia on the affected limb. Carrageenan treatment produced significant bilateral limb mechanical hyperalgesia 3 h post-injection. Up-regulation of mGluR 3 and mGluR 5 mRNA was observed in ipsilateral spinal cord recovered from clinically lame animals, restricted to laminae II–V and I–II, respectively. Western blot analyses of protein extracts revealed a bilateral increase in mGluR 2/3 and mGluR 5. No change was detected in spinal cord mGluR 1 or mGluR 2 mRNA. There was no change in mGluR 1,2,3,5 subtype mRNA or proteins in spinal cord recovered from animals 3 h post-carrageenan. These results demonstrate for the first time that mGluR subtypes are differentially expressed in spinal cord dorsal horn in response to persistent inflammation, and suggest that mGluR activity may be involved in mediating altered behaviours associated with clinical inflammatory pain.

Selective Loss of trans-Acting Instability Determinants of Neurofilament mRNA in Amyotrophic Lateral Sclerosis Spinal Cord

Neurofilament (NF) aggregates in motor neurons are a key neuropathological feature of amyotrophic lateral sclerosis (ALS). We have previously observed an alteration in the stoichiometry of NF subunit steady state mRNA levels in ALS spinal motor neurons using in situ hybridization and proposed that this led to aggregate formation. We have now examined the levels of NF mRNA in whole tissue homogenates of spinal cord using the RNase protection assay and real time reverse transcriptase-PCR and observed significant elevations of NF mRNA level in ALS. Compared with age-matched control, we observed a greater stability of heterogeneously expressed NFL mRNA in the presence of ALS spinal cord homogenates. Heat denaturing or protease K digestion of the control homogenates increased the stability of the NFL mRNA to levels observed in ALS homogenate. Increased NFL mRNA stability was also induced by increasing the percentage of ALS homogenate in an admixture of control and ALS homogenates. These observations suggest the presence of trans-acting NFL mRNA-destabilizing elements in control but not in ALS spinal cord homogenates. This was confirmed in gel retardation assays. We also observed that the destabilizing elements interact with the 3'-untranslated region of NFL mRNA. These findings suggest that the trans-acting NFL-destabilizing elements are selectively suppressed in ALS homogenates, resulting in an increased stability and level of NFL mRNA.

Functional characterization of prostaglandin F2alpha receptor in the spinal cord for tactile pain (allodynia).

Prostaglandin F2alpha (PGF2alpha) binds to its receptor (FP) to increase the intracellular-free calcium concentration ([Ca2+]i) by coupling of FP with Gq protein. Spinal intrathecal administration of PGF2alpha to mouse induces touch-evoked pain (mechanical allodynia), in which capsaicin-insensitive primary afferent Abeta-fibres and N-methyl-d-aspartate receptor epsilon 4 subunit are involved. FP in the spinal cord, however, was not well characterized. Here, we showed constitutive expression of FP mRNA in mouse spinal cord, and functionally characterized spinal FP-expressing cells which were involved in PGF2alpha-induced mechanical allodynia. The method for repetitive administration of oligodeoxyribonucleotides through tubing to conscious mice was established for mechanical allodynia evaluation. We identified an antisense oligodeoxyribonucleotide targeting FP mRNA, causing both disappearance of PGF2alpha-induced mechanical allodynia and decrease of FP mRNA. With saline-administered mice, PGF2alpha rapidly increased [Ca2+]i of the cells in the deeper layer of the dorsal horn. In contrast, when the FP antisense oligodeoxyribonucleotide was repeatedly administered, the population of PGF2alpha-responsive cells in the slices reduced, and PGF2alpha-induced [Ca2+]i increase of these cells diminished. These data strongly suggested that, in the dorsal horn of the spinal cord, there are the FP-expressing cells which are involved in PGF2alpha-induced mechanical allodynia.