Characterisation of peripheral and central components of the rat monoiodoacetate model of Osteoarthritis.

OBJECTIVE: Pain is the main reason patients report Osteoarthritis (OA), yet current analgesics remain relatively ineffective. This study investigated both peripheral and central mechanisms that lead to the development of OA associated chronic pain. DESIGN: The monoiodoacetate (MIA) model of OA was investigated at early (2-6 days post injection) and late (>14 days post injection) time points. Pain-like behaviour and knee histology were assessed to understand the extent of pain due to cartilage degradation. Electrophysiological single-unit recordings were taken from spinal wide dynamic range (WDR) neurons to investigate Diffuse Noxious Inhibitory Controls (DNIC) as a marker of potential changes in descending controls. Immunohistochemistry was performed on dorsal root ganglion (DRG) neurons to assess any MIA induced neuronal damage. Furthermore, qPCR was used to measure levels of glia cells and cytokines in the dorsal horn. RESULTS: Both MIA groups develop pain-like behaviour but only late phase (LP) animals display extensive cartilage degradation. Early phase animals have a normally functioning DNIC system but there is a loss of DNIC in LP animals. We found no evidence for neuronal damage caused by MIA in either group, yet an increase in IL-1ß mRNA in the dorsal horn of LP animals. CONCLUSION: The loss of DNIC in LP MIA animals suggests an imbalance in inhibitory and facilitatory descending controls, and a rise in the mRNA expression of IL-1ß mRNA suggest the development of central sensitisation. Therefore, the pain associated with OA in LP animals may not be attributed to purely peripheral mechanisms.

Pharmacology of reflex blinks in the rat: a novel model for headache research.

BACKGROUND: Migraineurs are highly sensitive to the nitric oxide donor glyceryl trinitrate which triggers attacks in many sufferers. In animal studies, glyceryl trinitrate increases neuronal activity in the trigeminovascular pathway and elevates neurotransmitter levels in the brainstem. Many migraineurs also display alterations in blink reflexes, known to involve brainstem circuits. We investigated the effect of GTN on evoked blinks in the anaesthetised rat to determine whether such reflexes may prove useful as the basis for a novel animal model to evaluate potential anti-migraine therapeutic agents. METHOD: In anaesthetised rats the electromyogram associated with the reflex blink evoked by corneal airpuff was recorded. Rats were infused with glyceryl trinitrate, sumatriptan plus glyceryl trinitrate or vehicle control. Changes in the magnitude of the reflex blink-associated electromyogram following these treatments were measured. RESULTS: Glyceryl trinitrate potentiated the evoked reflex blink-associated EMG response from 2 h after infusion. That effect was abolished by simultaneous infusion of sumatriptan with glyceryl trinitrate. CONCLUSIONS: These results show that simple skin surface measurements of evoked electromyographic activity in the rat can reliably detect the evoked blink reflex that can be potentiated by nitric oxide donors. This novel model may be an effective tool for evaluating putative anti-migraine therapeutic agents.

Macrophage-sensory neuronal interaction in HIV-1 gp120-induced neurotoxicity‡.

BACKGROUND: Human immunodeficiency virus (HIV)-associated sensory neuropathy (SN) is the most frequent neurological complication of HIV disease. Among the probable mechanisms underlying HIV-SN are neurotoxicity induced by the HIV glycoprotein gp120 and antiretroviral therapies (ART). Since HIV-SN prevalence remains high in patients who have not been exposed to toxic ART drugs, here we focused on gp120-mediated mechanisms underlying HIV-SN. METHODS: We hypothesized that a direct gp120-sensory neurone interaction is not the cause of neurite degeneration; rather, an indirect interaction of gp120 with sensory neurones involving macrophages underlies axonal degeneration. Rat dorsal root ganglion (DRG) cultures were used to assess gp120 neurotoxicity. Rat bone marrow-derived macrophage (BMDM) cultures and qPCR array were used to assess gp120-associated gene expression changes. RESULTS: gp120 induced significant, but latent onset, neurite degeneration until 24 h after application. gp120-neurone interaction occurred within 1 h of application in <10% of DRG neurones, despite neurite degeneration having a global effect. Application of culture media from gp120-exposed BMDMs induced a significant reduction in DRG neurite outgrowth. Furthermore, gp120 significantly increased the expression of 25 cytokine-related genes in primary BMDMs, some of which have been implicated in other painful polyneuropathies. The C-C chemokine receptor type 5 (CCR5) antagonist, maraviroc, concentration-dependently inhibited gp120-induced tumour necrosis factor-α gene expression, indicating that these effects occurred via gp120 activation of CCR5. CONCLUSIONS: Our findings highlight macrophages in the pathogenesis of HIV-SN and upstream modulation of macrophage response as a promising therapeutic strategy.

Efficient gene expression from integration-deficient lentiviral vectors in the spinal cord.

Gene transfer to spinal cord cells may be crucial for therapy in spinal muscular atrophy, amyotrophic lateral sclerosis and spinal cord injury. Lentiviral vectors are efficient for transduction of a variety of cells, but like all integrating vectors they pose a risk of insertional mutagenesis. Integration-deficient lentiviral vectors (IDLVs) remain episomal but retain the transduction efficiency of standard integrating lentiviral vectors, particularly when the episomes are not diluted out through repeated cell division. We have now applied IDLVs for transduction of spinal cord in vitro, in explants and in vivo. Our results demonstrate similar efficiency of eGFP expression from integrating lentiviral vectors and IDLVs in most cell types analyzed, including motor neurons, interneurons, dorsal root ganglia (DRG) neurons and astroglia. IDLV-mediated expression of pro-glial-cell-derived neurotrophic factor (Gdnf) rescues motor neuron cultures from death caused by removal of exogenous trophic support. IDLVs also mediate efficient RNA interference in DRG neuron cultures. After intraparenchymal injection in the rat and mouse cervical and lumbar regions in vivo, transduction is mainly neuronal, with both motor neurons and interneurons being efficiently targeted. These results suggest that IDLVs could be efficient and safer tools for spinal cord transduction in future therapeutic strategies.

Genomics of pain in osteoarthritis.

Osteoarthritis (OA) accounts for the majority of the disease burden for musculoskeletal disorders and is one of the leading causes of disability worldwide. This disability is the result not of the cartilage loss that defines OA radiographically, but of the chronic pain whose presence defines symptomatic OA. It is becoming clear that many genes, each with a small effect size, contribute to the risk of developing OA. However, the genetics of OA pain are only just starting to be explored. This review will describe the first genes to have been identified in genomic studies of OA pain, as well as the possible dual roles of genes previously identified in genomic studies of OA in the context of pain. Difficulties associated with attempting to characterise the genetics of OA pain will be discussed and promising future avenues of research into genetic and epigenetic factors affecting OA pain described.

Targeting novel peripheral mediators for the treatment of chronic pain.

Research efforts over the past two decades have helped us better understand the biological mechanisms that lead to chronic pain. Despite this, there has been limited progress in developing novel analgesics to treat sufferers of persistent pain conditions, who may account for as many as one-fifth of the population. A re-evaluation of the strategies used to discover pain-relieving drugs is needed to meet this widespread clinical need. Here, we discuss the merits of pursuing peripherally acting pain mediators. We review the significant clinical evidence that neuronal activity from the periphery is a major contributor to painful symptom production and that peripheral mediators play a substantial role in this aberrant nociceptor activity. We discuss the clinical benefits of blocking individual known mediators and describe our own approach to identify novel mediators.

Interstitial cystitis/bladder pain syndrome: diagnosis and management.

INTRODUCTION AND HYPOTHESIS: The bladder pain syndrome (BPS) is a spectrum of urological symptoms characterised by bladder pain with typical cystoscopic features. Diagnosis and management of this syndrome may be difficult. There is no evidence-based management approach for the diagnosis or treatment of BPS. The objective of this study was to critically review and summarise the evidence relating to the diagnosis and treatment of the bladder pain syndrome. METHODS: A review of published data on the diagnosis and treatment of the BPS was performed. Our search was limited to English-language articles, on the "diagnosis", and "management" or "treatment" of "interstitial cystitis" and the "bladder pain syndrome" in "humans." RESULTS: Frequency, urgency and pain on bladder filling are the most common symptoms of BPS. All urodynamic volumes are reduced in patients with BPS. Associated conditions include psychological distress, depression, history of sexual assault, irritable bowel syndrome and fibromyalgia. Cystoscopy remains the test for definitive diagnosis, with visualisation of haemorrhage on cystoreduction. A multidisciplinary treatment approach is essential in the management of this condition. Orally administered amitriptyline is an efficacious medical treatment for BPS. Intravesical hyaluronic acid and local anaesthetic, with/without hydrodistension are among new treatment strategies. Sacral or pudendal neuromodulation is effective, minimally invasive and safe. Surgery is reserved for refractory cases. CONCLUSIONS: There remains a paucity of evidence for the diagnosis and treatment of BPS. We encountered significant heterogeneity in the assessment of symptoms, duration of treatment and follow up of patients in our literature review.

The biological effects of endogenous nerve growth factor on adult sensory neurons revealed by a trkA-IgG fusion molecule.

Evidence suggests that nerve growth factor (NGF) may function as a mediator of some persistent pain states. We have used a synthetic protein, trkA-IgG, to sequester endogenous NGF and block the survival effects of NGF on cultured sensory neurons. We show that administration of trkA-IgG produces a sustained thermal and chemical hypoalgesia and leads to a downregulation of the sensory neuropeptide CGRP (calcitonin gene-related peptide) in treated sensory neurons. Acute administration of the molecule blocks the hyperalgesia that develops with carrageenan-induced inflammation. These data suggest that peripherally produced NGF normally acts to maintain the sensitivity of nociceptive sensory neurons and that, in some inflammatory states, an upregulation of NGF is responsible for alterations in pain-related behaviour. Antagonists of NGF may therefore be of clinical use in treating some chronic pain states.

Activation of the p21ras pathway couples antigen receptor stimulation to induction of the primary response gene egr-1 in B lymphocytes.

The primary response gene egr-1 encodes a sequence-specific transcription factor whose expression is necessary for antigen receptor-stimulated activation of B lymphocytes. The molecular processes involved in linking egr-1 induction to antigen receptor signaling have not been defined. The present study demonstrates that expression of an activated form of p21ras results in egr-1 induction similar to that previously shown after antigen receptor cross-linking. In addition, both antigen receptor cross-linking and p21ras use the same element in the egr-1 promoter to exert their effects. Using dominant-negative mutants of p21ras and raf-1, we demonstrate that induction of egr-1 after antigen receptor cross-linking is mediated by activation of the p21ras/mitogen-activated protein kinase signaling pathway. While regulation of the p21ras pathway during B cell activation has been intensively studied, this report represents the first description of a biologically relevant event associated with its activation.

Potent analgesic effects of GDNF in neuropathic pain states.

Neuropathic pain arises as a debilitating consequence of nerve injury. The etiology of such pain is poorly understood, and existing treatment is largely ineffective. We demonstrate here that glial cell line-derived neurotrophic factor (GDNF) both prevented and reversed sensory abnormalities that developed in neuropathic pain models, without affecting pain-related behavior in normal animals. GDNF reduces ectopic discharges within sensory neurons after nerve injury. This may arise as a consequence of the reversal by GDNF of the injury-induced plasticity of several sodium channel subunits. Together these findings provide a rational basis for the use of GDNF as a therapeutic treatment for neuropathic pain states.

Methylation of an immediate-early inducible gene as a mechanism for B cell tolerance induction.

Stage-specific gene regulation is important in determining cell function during development. Immature B cells expressing membrane-bound immunoglobulin M (mIgM) are sensitive to antigen-induced tolerance, whereas mature B cells are activated by antigen. Previous studies have established an association between Egr-1 gene induction and antigen receptor (mIgM)-mediated activation of mature B cells. Here it is shown that the immature B cell line WEHI-231 and tolerance-sensitive bone marrow-derived B cells do not express Egr-1. It is further shown that lack of inducible expression in these cells is due to specific methylation of the Egr-1 gene. Thus, covalent inactivation of an activation-associated gene may explain tolerance sensitivity at specific stages of B cell development.

Expression and coexpression of Trk receptors in subpopulations of adult primary sensory neurons projecting to identified peripheral targets.

To determine whether neurotrophins act on functionally distinct populations of adult sensory neurons, the distributions of mRNAs for TrkA and tyrosine kinase-containing isoforms of TrkB and TrkC were determined in rat DRG neurons projecting to different peripheral targets. Whereas trkA was expressed by a very high percentage of visceral afferents, trkC was expressed frequently only in muscle afferents. Among cutaneous afferents, the size distributions for trkA- and trkC-positive cells showed little overlap. The percentages and size distributions of cells labeled for the trks argue strongly that almost all trkB-expressing cells must also express trkA or trkC. These results indicate that NGF and NT-3 act on functionally distinct populations of adult sensory neurons and suggest that a sizeable number of small DRG neurons may not respond to neurotrophins via a known Trk in the adult rat.

Schwann cell precursors transplanted into the injured spinal cord multiply, integrate and are permissive for axon growth.

There is a strong current interest in the use of cell transplantation for the treatment of spinal cord injuries. We report here the novel and potentially useful properties of an early cell in the Schwann cell lineage, the Schwann cell precursor (SCP). The experiments reveal a striking difference between these cells and Schwann cells when transplanted into the CNS. Unlike Schwann cells, SCPs thrive in the CNS where they initially proliferate rapidly but then fall out of division, thus effectively filling up the large cystic cavities formed following crush injury, while avoiding tumor formation. By 8 weeks, SCPs had started to express S100beta protein, a marker that differentiates Schwann cells from SCPs and had formed an apparently stable, vascularized cell mass, which created a continuous cellular bridge across the cystic cavities. The formation of the surrounding glial scar was reduced by local spread of the transplanted cells into the surrounding CNS tissue, where the cells integrated intimately with astrocytes and attenuated the physical barrier they normally form. SCP transplantation also altered and reduced the expression of chondroitin sulfate proteoglycans around the injury site. Caudal to the SCP transplants there was a large increase in the number of axons, compared with that seen in nontransplanted control tissue, showing that the implants effectively support axonal growth or sprouting. SCPs have advantageous attributes for CNS repair, despite the fact that sticky tape removal and ladder crossing tests at 8 weeks did not reveal significant functional improvements when compared with control animals.

The tetrodotoxin-resistant sodium channel SNS has a specialized function in pain pathways.

Many damage-sensing neurons express tetrodotoxin (TTX)-resistant voltage-gated sodium channels. Here we examined the role of the sensory-neuron-specific (SNS) TTX-resistant sodium channel alpha subunit in nociception and pain by constructing sns-null mutant mice. These mice expressed only TTX-sensitive sodium currents on step depolarizations from normal resting potentials, showing that all slow TTX-resistant currents are encoded by the sns gene. Null mutants were viable, fertile and apparently normal, although lowered thresholds of electrical activation of C-fibers and increased current densities of TTX-sensitive channels demonstrated compensatory upregulation of TTX-sensitive currents in sensory neurons. Behavioral studies demonstrated a pronounced analgesia to noxious mechanical stimuli, small deficits in noxious thermoreception and delayed development of inflammatory hyperalgesia. These data show that SNS is involved in pain pathways and suggest that blockade of SNS expression or function may produce analgesia without side effects.

Preoperative ultraviolet B inflammation in skin: Modelling individual differences in acute postoperative pain and neuro-immune interactions.

BACKGROUND: Neuroimmune interactions play a vital role in many of the most common pain conditions, such as arthritis. There have been many attempts to derive clinically predictive information from an individual's inflammatory response in order to gauge subsequent pain perception. OBJECTIVES: Here, we wanted to test whether this effort could be enhanced and complemented by the use of a model system which takes into account the function of not just circulating, but also tissue-resident immune cells: ultraviolet B (UVB) irradiation of the skin. METHODS: We conducted psychophysical and transcriptional analysis of hyperalgesia arising as a result of UVB-induced inflammation in patients before total knee arthroplasty (TKA, n = 23). Levels of acute postoperative pain were assessed and correlated with preoperative data. RESULTS: Cytokine and chemokine responses after UVB irradiation were found to be inversely correlated with the level of pain experienced after surgery (Spearman's ρ = -0.498). CONCLUSION: It may be possible to use this simple model to study and predict the nature of neuro-immune responses at more remote, clinically relevant sites. SIGNIFICANCE: A simple model of UVB inflammation in the skin might predict the degree of a patient's neuro-immune response and the extent of their postoperative pain after total knee arthroplasty.

Chondroitinase ABC promotes sprouting of intact and injured spinal systems after spinal cord injury.

Chondroitin sulfate proteoglycans (CSPGs) are inhibitory extracellular matrix molecules that are upregulated after CNS injury. Degradation of CSPGs using the enzyme chondroitinase ABC (ChABC) can promote functional recovery after spinal cord injury. However, the mechanisms underlying this recovery are not clear. Here we investigated the effects of ChABC treatment on promoting plasticity within the spinal cord. We found robust sprouting of both injured (corticospinal) and intact (serotonergic) descending projections as well as uninjured primary afferents after a cervical dorsal column injury and ChABC treatment. Sprouting fibers were observed in aberrant locations in degenerating white matter proximal to the injury in regions where CSPGs had been degraded. Corticospinal and serotonergic sprouting fibers were also observed in spinal gray matter at and below the level of the lesion, indicating increased innervation in the terminal regions of descending projections important for locomotion. Spinal-injured animals treated with a vehicle solution showed no significant sprouting. Interestingly, ChABC treatment in uninjured animals did not induce sprouting in any system. Thus, both denervation and CSPG degradation were required to promote sprouting within the spinal cord. We also examined potential detrimental effects of ChABC-induced plasticity. However, although primary afferent sprouting was observed after lumbar dorsal column lesions and ChABC treatment, there was no increased connectivity of nociceptive neurons or development of mechanical allodynia or thermal hyperalgesia. Thus, CSPG digestion promotes robust sprouting of spinal projections in degenerating and denervated areas of the spinal cord; compensatory sprouting of descending systems could be a key mechanism underlying functional recovery.

A ternary complex factor-dependent mechanism mediates induction of egr-1 through selective serum response elements following antigen receptor cross-linking in B lymphocytes.

Induction of the primary response gene egr-1 occurs rapidly following antigen receptor cross-linking in B lymphocytes. Antisense studies have demonstrated that this induction is necessary for their subsequent activation to this signal. The present study examines the molecular mechanism whereby the receptor-generated signals interact with the egr-1 promoter to elicit transcription. Deletion mapping and point mutations have indicated that two of the five serum response elements (SREs) in the egr-1 promoter can mediate induction. Of the two critical SREs, both are capable of mediating maximal induction even in the absence of the other SRE. Our results also indicate that adjacent Ets motifs are necessary for induction. Like the c-fos SRE, the egr-1 SRE/Ets sites are occupied by a multiprotein (ternary) complex containing a homodimer of serum response factor and an unidentified member of the Ets family of transcription factors. The identification of a ternary complex-dependent mechanism of egr-1 induction, along with selective utilization of SREs in B lymphocytes, suggests that a complicated array of signaling cascades interacts with unique combinations of regulatory elements in the egr-1 promoter in different cell types.

The essential cofactor TRRAP recruits the histone acetyltransferase hGCN5 to c-Myc.

The c-Myc protein functions as a transcription factor to facilitate oncogenic transformation; however, the biochemical and genetic pathways leading to transformation remain undefined. We demonstrate here that the recently described c-Myc cofactor TRRAP recruits histone acetylase activity, which is catalyzed by the human GCN5 protein. Since c-Myc function is inhibited by recruitment of histone deacetylase activity through Mad family proteins, these opposing biochemical activities are likely to be responsible for the antagonistic biological effects of c-Myc and Mad on target genes and ultimately on cellular transformation.

Itching for an explanation.

Itch is a distinct sensation arising from the superficial layers of skin and mucous membranes. It is elicited by histamine and probably other endogenous chemicals that excite subpopulations of unmyelinated primary afferents and spinal neurones projecting through the anterolateral quadrant to the brain. The two popular views, which propose either that itch is signalled by a labelled line system of peripheral and central itch-specific neurones or that itch is the subliminal form of pain, both fail to explain convincingly many known features. Alternative theories emphasize central processes that extract the relevant information from afferents with broad sensitivity spectra for pruritogenic and noxious stimuli. Thus, itch presents an irritating challenge for the specificity theory of somatosensation.

Does the right side know what the left is doing?

Following peripheral-nerve lesions there are well-documented events that affect the contralateral nonlesioned structures. These contralateral effects are qualitatively similar to those occurring at the ipsilateral side, but are usually smaller in magnitude and have a briefer time course. It is unclear whether the findings are an epiphenomenon or serve a biological purpose, but in either case the existence of these effects implies the presence of unrecognized signalling mechanisms that link the two sides of the body. Strong circumstantial evidence argues against a peripheral mechanism (for example, via circulating factors) and in favour of a central mechanism, in particular signalling via the system of commissural interneurons that is present in spinal cord and brainstem. While an altered pattern of activity in this system might underlie the phenomenon, there are several reasons for proposing that the changes depend upon chemical signals, possibly growth factors. Because of its relative easy access for experimental manipulation, the spinal cord could serve as a model system to study these transmedian signalling systems.