Corrigendum to "Single-chain fragment variable antibody targeting cholecystokinin-B receptor for pain reduction" [Neurobiol. Pain 10 (2021) 100067].

[This corrects the article DOI: 10.1016/j.ynpai.2021.100067.].

Single-chain Fragment variable antibody targeting cholecystokinin-B receptor for pain reduction.

The cholecystokinin B receptor and its neuropeptide ligand are upregulated in chronic neuropathic pain models. Single-chain Fragment variable antibodies were generated as preferred non-opioid targeting therapy blocking the cholecystokinin B receptor to inhibit chronic neuropathic pain models in vivo and in vitro. Engineered antibodies of this type feature binding activity similar to monoclonal antibodies but with stronger affinity and increased tissue penetrability due to their smaller size. More importantly, single-chain Fragment variable antibodies have promising biotherapeutic applications for both nervous and immune systems, now recognized as interactive in chronic pain. A mouse single-chain Fragment variable antibody library recognizing a fifteen amino acid extracellular peptide fragment of the cholecystokinin B receptor was generated from immunized spleens. Ribosome display, a powerful cell-free technology, was applied for recombinant antibody selection. Antibodies with higher affinity, stability, solubility, and binding specificity for cholecystokinin B not A receptor were selected and optimized for in vivo and in vitro efficacy. A single dose of the lead candidate reduced mechanical and cold hypersensitivity in two rodent models of neuropathic pain for at least seven weeks. Continuing efficacy was evident with either intraperitoneal or intranasal dosing. Likewise, the lead single-chain Fragment variable antibody totally prevented development of anxiety- and depression-like behaviors and cognitive deficits typical in the models. Reduction of neuronal firing frequency was evident in trigeminal ganglia primary neuronal cultures treated in vitro with the cholecystokinin B receptor antibody. Immunofluorescent staining intensity in the trigeminal neuron primary cultures was significantly reduced incrementally after overnight binding with increasingly higher dilutions of the single-chain Fragment variable antibody. While it is reported that single-chain Fragment variable antibodies are removed systemically within 2-6 h, Western blot evidence indicates the His-tag marker remained after 7 weeks in the trigeminal ganglia and in the dorsolateral medulla, providing evidence of brain and ganglia penetrance known to be compromised in overactivated states. This project showcases the in vivo efficacy of our lead single-chain Fragment variable antibody indicating its potential for development as a non-opioid, non-addictive therapeutic intervention for chronic pain. Importantly, studies by others have indicated treatments with cholecystokinin B receptor antagonists suppress maintenance and reactivation of morphine dependence in place preference tests while lowering tolerance and dose requirements. Our future studies remain to address these potential benefits that may accompany the cholecystokinin B receptor biological therapy. Both chronic sciatic and orofacial pain can be unrelenting and excruciating, reducing quality of life as well as diminishing physical and mental function. An effective non-opiate, non-addictive therapy with potential to significantly reduce chronic neuropathic pain long term is greatly needed.

Inflammatory 'double hit' model of temporomandibular joint disorder with elevated CCL2, CXCL9, CXCL10, RANTES and behavioural hypersensitivity in TNFR1/R2-/- mice.

BACKGROUND: Patients with temporomandibular joint disorders (TMD), reactive arthritis and rheumatoid arthritis often have combined etiology of hereditary and microenvironmental factors contributing to joint pain. Multiple clinical and animal studies indicate 'double-hit' inflammatory insults can cause chronic inflammation. The first inflammatory insult primes the immune system and subsequent insults elicit amplified responses. The present 'double hit' study produced a chronic orofacial pain model in mice with genetic deletion of both TNFα receptors (TNFR1/R2-/-), investigating the main nociceptive signalling pathways in comparisons to wild type mice. METHODS: An initial inflammatory insult was given unilaterally into the temporomandibular joint (TMJ). Secondary hypersensitivity was tested on the skin over the TMJ throughout the experiment. Three weeks later after complete reversal of hypersensitivity, a second inflammatory insult was imposed on the colon. Pharmacological interventions were tested for efficacy after week 10 when hypersensitivity was chronic in TNFR1/R2-/- mice. Serum cytokines were analysed at Days 1, 14, and Week 18. RESULTS: The double hit insult produced chronic hypersensitivity continuing through the 4-month experimental timeline in the absence of TNFα signalling. P2X7 and NMDA receptor antagonists temporarily attenuated chronic hypersensitivity. Serum cytokine/chemokine analysis on Day 14 when CFA induced hypersensitivity was resolved identified increased levels of pro-inflammatory cytokines CCL2, CXCL9, CXCL10, RANTES and decreased levels of anti-inflammatory cytokines IL-1ra and IL-4 in TNFR1/R2-/- compared to WT mice. CONCLUSIONS: These data suggest a causal feed-forward signalling cascade of these little studied cytokines have the potential to cause recrudescence in this orofacial inflammatory pain model in the absence of TNFα signalling. SIGNIFICANCE: Using a mouse model of chronic inflammatory temporomandibular joint disorder, we determined that absence of functional TNFR1/R2 induces aberrant inflammatory signalling caused by other increased pro-inflammatory and decreased anti-inflammatory cytokines that could serve as blood biomarkers and may predict disease progression.

A Mouse Model of Chronic Pancreatitis Induced by an Alcohol and High Fat Diet.

BACKGROUND/AIMS: Study of acute pancreatitis in chemically-induced rodent models has provided useful data; models of alcoholic chronic pancreatitis have not been available in mice. The aim of the present study was to characterize a mouse model of chronic pancreatitis induced solely with an alcohol and high fat (AHF) diet. METHODS: Mice were fed a liquid high fat diet containing 6% alcohol as well as a high fat supplement (57% total dietary fat) over a period of five months or as control, normal chow ad libitum. Pain related measures utilized as an index of pain included mechanical sensitivity of the hind paws determined using von Frey filaments and a smooth/rough textured plate. A modified hotplate test contributed information about higher order behavioral responses to visceral hypersensitivity. Mice underwent mechanical and thermal testing both with and without pharmacological treatment with a peripherally restricted µ-opioid receptor agonist, loperamide. RESULTS: Mice on the AHF diet exhibited mechanical and heat hypersensitivity as well as fibrotic histology indicative of chronic pancreatitis. Low dose, peripherally restricted opiate loperamide attenuated both mechanical and heat hypersensitivity. CONCLUSION: Mice fed an alcohol and high fat diet develop histology consistent with chronic pancreatitis as well as opioid sensitive mechanical and heat hypersensitivity.

GABA-A receptor activity in the noradrenergic locus coeruleus drives trigeminal neuropathic pain in the rat; contribution of NAα1 receptors in the medial prefrontal cortex.

Trigeminal neuropathic pain is described as constant excruciating facial pain. The study goal was to investigate the role of nucleus locus coeruleus (LC) in a model of chronic orofacial neuropathic pain (CCI-ION). The study examines LC's relationship to both the medullary dorsal horn receiving trigeminal nerve sensory innervation and the medial prefrontal cortex (mPFC). LC is a major source of CNS noradrenaline (NA) and a primary nucleus involved in pain modulation. Although descending inhibition of acute pain by LC is well established, contribution of the LC to facilitation of chronic neuropathic pain is also reported. In the present study, a rat orofacial pain model of trigeminal neuropathy was induced by chronic constrictive injury of the infraorbital nerve (CCI-ION). Orofacial neuropathic pain was indicated by development of whisker pad mechanical hypersensitivity. Hypersensitivity was alleviated by selective elimination of NA neurons, including LC (A6 cell group), with the neurotoxin anti-dopamine-ß-hydroxylase saporin (anti-DßH-saporin) microinjected either intracerebroventricularly (i.c.v.) or into trigeminal spinal nucleus caudalis (spVc). The GABAA receptor antagonist, bicuculline, administered directly into LC (week 8) inhibited hypersensitivity. This indicates a valence shift in which increased GABAA signaling ongoing in LC after trigeminal nerve injury paradoxically produces excitatory facilitation of the chronic pain state. Microinjection of NAα1 receptor antagonist, benoxathian, into mPFC attenuated whisker pad hypersensitivity, while NAα2 receptor antagonist, idazoxan, was ineffective. Thus, GABAA-mediated activation of NA neurons during CCI-ION can facilitate hypersensitivity through NAα1 receptors in the mPFC. These data indicate LC is a chronic pain generator.

Transdermal cannabidiol reduces inflammation and pain-related behaviours in a rat model of arthritis.

BACKGROUND: Current arthritis treatments often have side-effects attributable to active compounds as well as route of administration. Cannabidiol (CBD) attenuates inflammation and pain without side-effects, but CBD is hydrophobic and has poor oral bioavailability. Topical drug application avoids gastrointestinal administration, first pass metabolism, providing more constant plasma levels. METHODS: This study examined efficacy of transdermal CBD for reduction in inflammation and pain, assessing any adverse effects in a rat complete Freund's adjuvant-induced monoarthritic knee joint model. CBD gels (0.6, 3.1, 6.2 or 62.3 mg/day) were applied for 4 consecutive days after arthritis induction. Joint circumference and immune cell invasion in histological sections were measured to indicate level of inflammation. Paw withdrawal latency (PWL) in response to noxious heat stimulation determined nociceptive sensitization, and exploratory behaviour ascertained animal's activity level. RESULTS: Measurement of plasma CBD concentration provided by transdermal absorption revealed linearity with 0.6-6.2 mg/day doses. Transdermal CBD gel significantly reduced joint swelling, limb posture scores as a rating of spontaneous pain, immune cell infiltration and thickening of the synovial membrane in a dose-dependent manner. PWL recovered to near baseline level. Immunohistochemical analysis of spinal cord (CGRP, OX42) and dorsal root ganglia (TNFα) revealed dose-dependent reductions of pro-inflammatory biomarkers. Results showed 6.2 and 62 mg/day were effective doses. Exploratory behaviour was not altered by CBD indicating limited effect on higher brain function. CONCLUSIONS: These data indicate that topical CBD application has therapeutic potential for relief of arthritis pain-related behaviours and inflammation without evident side-effects.

Alcohol and high fat induced chronic pancreatitis: TRPV4 antagonist reduces hypersensitivity.

The pathogenesis of pain in chronic pancreatitis is poorly understood, and its treatment can be a major clinical challenge. Surgical and other invasive methods have variable outcomes that can be unsatisfactory. Therefore, there is a great need for further discovery of the pathogenesis of pancreatitis pain and new therapeutic targets. Human and animal studies indicate a critical role for oxidative stress and activation of transient receptor potential (TRP) cation channel subfamily members TRPV1 and TRPA1 on pancreatic nociceptors in sensitization mechanisms that result in pain. However, the in vivo role of transient receptor potential cation channel subfamily V member 4 (TRPV4) in chronic pancreatitis needs further evaluation. The present study characterized a rat alcohol/high fat diet (AHF)-induced chronic pancreatitis model with hypersensitivity, fibrotic pathology, and fat vacuolization consistent with the clinical syndrome. The rats with AHF-induced pancreatitis develop referred visceral pain-like behaviors, i.e. decreased hindpaw mechanical thresholds and shortened abdominal and hindpaw withdrawal latency to heat. In this study, oxidative stress was characterized as well as the role of TRPV4 in chronic visceral hypersensitivity. Lipid peroxidase and oxidative stress were indicated by increased plasma thiobarbituric acid reactive substances (TBARS) and diminished pancreatic manganese superoxide dismutase (MnSOD). The secondary sensitization associated with AHF-induced pancreatitis was effectively alleviated by the TRPV4 antagonist, HC 067047. Similarity of the results to those with the peripherally restricted µ-opiate receptor agonist, loperamide, suggested TRPV4 channel activated peripheral sensitization. This study using a reliable model that provides pre-clinical correlates of human chronic pancreatitis provides further evidence that TRPV4 channel is a potential therapeutic target for treatment of pancreatitis pain.

Dysregulated TNFα promotes cytokine proteome profile increases and bilateral orofacial hypersensitivity.

BACKGROUND: Tumor necrosis factor alpha (TNFα) is increased in patients with headache, neuropathic pain, periodontal and temporomandibular disease. This study and others have utilized TNF receptor 1/2 (TNFR1/2) knockout (KO) animals to investigate the effect of TNFα dysregulation in generation and maintenance of chronic neuropathic pain. The present study determined the impact of TNFα dysregulation in a trigeminal inflammatory compression (TIC) nerve injury model comparing wild-type (WT) and TNFR1/2 KO mice. METHODS: Chromic gut suture was inserted adjacent to the infraorbital nerve to induce the TIC model mechanical hypersensitivity. Cytokine proteome profiles demonstrated serology, and morphology explored microglial activation in trigeminal nucleus 10weeks post. RESULTS: TIC injury induced ipsilateral whisker pad mechanical allodynia persisting throughout the 10-week study in both TNFR1/2 KO and WT mice. Delayed mechanical allodynia developed on the contralateral whisker pad in TNFR1/2 KO mice but not in WT mice. Proteomic profiling 10weeks after chronic TIC injury revealed TNFα, interleukin-1alpha (IL-1α), interleukin-5 (IL-5), interleukin-23 (IL-23), macrophage inflammatory protein-1ß (MIP-1ß), and granulocyte-macrophage colony-stimulating factor (GM-CSF) were increased more than 2-fold in TNFR1/2 KO mice compared to WT mice with TIC. Bilateral microglial activation in spinal trigeminal nucleus was detected only in TNFR1/2 KO mice. p38 mitogen-activated protein kinase (MAPK) inhibitor and microglial inhibitor minocycline reduced hypersensitivity. CONCLUSIONS: The results suggest the dysregulated serum cytokine proteome profile and bilateral spinal trigeminal nucleus microglial activation are contributory to the bilateral mechanical hypersensitization in this chronic trigeminal neuropathic pain model in the mice with TNFα dysregulation. Data support involvement of both neurogenic and humoral influences in chronic neuropathic pain.

Trigeminal Inflammatory Compression (TIC) injury induces chronic facial pain and susceptibility to anxiety-related behaviors.

Our laboratory previously developed a novel neuropathic and inflammatory facial pain model for mice referred to as the Trigeminal Inflammatory Compression (TIC) model. Rather than inducing whole nerve ischemia and neuronal loss, this injury induces only slight peripheral nerve demyelination triggering long-term mechanical allodynia and cold hypersensitivity on the ipsilateral whisker pad. The aim of the present study is to further characterize the phenotype of the TIC injury model using specific behavioral assays (i.e. light-dark box, open field exploratory activity, and elevated plus maze) to explore pain- and anxiety-like behaviors associated with this model. Our findings determined that the TIC injury produces hypersensitivity 100% of the time after surgery that persists at least 21 weeks post injury (until the animals are euthanized). Three receptive field sensitivity pattern variations in mice with TIC injury are specified. Animals with TIC injury begin displaying anxiety-like behavior in the light-dark box preference and open field exploratory tests at week eight post injury as compared to sham and naïve animals. Panic anxiety-like behavior was shown in the elevated plus maze in mice with TIC injury if the test was preceded with acoustic startle. Thus, in addition to mechanical and cold hypersensitivity, the present study identified significant anxiety-like behaviors in mice with TIC injury resembling the clinical symptomatology and psychosocial impairments of patients with chronic facial pain. Overall, the TIC injury model's chronicity, reproducibility, and reliability in producing pain- and anxiety-like behaviors demonstrate its usefulness as a chronic neuropathic facial pain model.

Catechol-O-methyltransferase inhibition alters pain and anxiety-related volitional behaviors through activation of ß-adrenergic receptors in the rat.

Reduced catechol-O-methyltransferase (COMT) activity resulting from genetic variation or pharmacological depletion results in enhanced pain perception in humans and nociceptive behaviors in animals. Using phasic mechanical and thermal reflex tests (e.g. von Frey, Hargreaves), recent studies show that acute COMT-dependent pain in rats is mediated by ß-adrenergic receptors (ßARs). In order to more closely mimic the characteristics of human chronic pain conditions associated with prolonged reductions in COMT, the present study sought to determine volitional pain-related and anxiety-like behavioral responses following sustained as well as acute COMT inhibition using an operant 10-45°C thermal place preference task and a light/dark preference test. In addition, we sought to evaluate the effects of sustained COMT inhibition on generalized body pain by measuring tactile sensory thresholds of the abdominal region. Results demonstrated that acute and sustained administration of the COMT inhibitor OR486 increased pain behavior in response to thermal heat. Further, sustained administration of OR486 increased anxiety behavior in response to bright light, as well as abdominal mechanosensation. Finally, all pain-related behaviors were blocked by the non-selective ßAR antagonist propranolol. Collectively, these findings provide the first evidence that stimulation of ßARs following acute or chronic COMT inhibition drives cognitive-affective behaviors associated with heightened pain that affects multiple body sites.

A rat knockout model implicates TRPC4 in visceral pain sensation.

Acute and chronic pain resulting from injury, surgery, or disease afflicts >100 million Americans each year, having a severe impact on mood, mental health, and quality of life. The lack of structural and functional information for most ion channels, many of which play key roles in the detection and transmission of noxious stimuli, means that there remain unidentified therapeutic targets for pain management. This study focuses on the transient receptor potential canonical subfamily 4 (TRPC4) ion channel, which is involved in the tissue-specific and stimulus-dependent regulation of intracellular Ca²âº signaling. Rats with a transposon-mediated TRPC4-knockout mutation displayed tolerance to visceral pain induced by colonic mustard oil (MO) exposure, but not somatic or neuropathic pain stimuli. Moreover, wild-type rats treated with a selective TRPC4 antagonist (ML-204) prior to MO exposure mimicked the behavioral responses observed in TRPC4-knockout rats. Significantly, ML-204 inhibited visceral pain-related behavior in a dose-dependent manner without noticeable adverse effects. These data provide evidence that TRPC4 is required for detection and/or transmission of colonic MO visceral pain sensation. In the future, inhibitors of TRPC4 signaling may provide a highly promising path for the development of first-in-class therapeutics for this visceral pain, which may have fewer side effects and less addictive potential than opioid derivatives.

Pine oil effects on chemical and thermal injury in mice and cultured mouse dorsal root ganglion neurons.

A commercial resin-based pine oil (PO) derived from Pinus palustris and Pinus elliottii was the major focus of this investigation. Extracts of pine resins, needles, and bark are folk medicines commonly used to treat skin ailments, including burns. The American Burn Association estimates that 500,000 people with burn injuries receive medical treatment each year; one-half of US burn victims are children, most with scald burns. This systematic study was initiated as follow-up to personal anecdotal evidence acquired over more than 10 years by MH Bhattacharyya regarding PO's efficacy for treating burns. The results demonstrate that PO counteracted dermal inflammation in both a mouse ear model of contact irritant-induced dermal inflammation and a second degree scald burn to the mouse paw. Furthermore, PO significantly counteracted the tactile allodynia and soft tissue injury caused by the scald burn. In mouse dorsal root ganglion neuronal cultures, PO added to the medium blocked adenosine triphosphate-activated, but not capsaicin-activated, pain pathways, demonstrating specificity. These results together support the hypothesis that a pine-oil-based treatment can be developed to provide effective in-home care for second degree burns.

Prolonged high fat/alcohol exposure increases TRPV4 and its functional responses in pancreatic stellate cells.

The present study investigated transient receptor potential vanilloid type 4 (TRPV4) ion channels in pancreatic stellate cells (PSCs) isolated from rats with high-fat and alcohol diet (HFA)-induced chronic pancreatitis. TRPV4 is a calcium-permeable nonselective ion channel responsive to osmotic changes, alcohol metabolites arachidonic acid, anandamide, their derivatives, and injury-related lipid mediators. Male Lewis rats were fed HFA for 6-8 wk before isolation and primary culture of PSCs. Control PSCs were harvested from rats fed standard chow. Immunoreactivity for cytoskeletal protein activation product α-smooth muscle actin (α-SMA) and platelet-derived growth factor receptor-ß subunit (PDGFR-ß) characterized the cells as PSCs. TRPV4 expression increased in PSCs of HFA-fed rats and control cultures after alcohol treatment (50 mM). Cell responses to activation of inducible TRPV4 were assessed with live cell calcium imaging. Threefold increased and sustained intracellular calcium mobilization responses occurred in 70% of pancreatic stellate cells from HFA-fed rats in response to TRPV4 activators arachidonic acid, lipid second messenger, phorbol ester 4 α-phorbol 12,13-didecanoate (4αPDD), and 50% hypoosmotic media compared with relatively unresponsive PSCs from control rats. Activation responses were attenuated by nonselective TRPV channel blocker ruthenium red. Tumor necrosis factor-α (TNF-α, 1 ng/ml, 16 h) increased responses to 4αPDD in control PSCs. These findings implicate TRPV4-mediated calcium responses inducible after HFA exposure and inflammation in reactive responses of activated PSCs that impair pancreatic function, such as responsiveness to cytokines and the deposition of collagen fibrosis that precipitates ductal blockage and pain.

Gene therapy for pancreatitis pain.

Pancreatic cancer and chronic pancreatitis are clinical syndromes associated with severe pain that is difficult to manage. Thus, seeking additional pain reduction therapies is warranted. Excessive alcohol consumption over an extended period of time is the primary causal agent in pancreatitis. The efficacy of a replication defective Herpes (HSV-1, DPE) viral vector construct encoding the human preproenkephalin gene (HSV-Enk), used as a molecular therapy for alleviation of pancreatitis pain, is reviewed here. The characteristics of the gene therapy treatment for inflammation and pain-related behavior in two alcoholic pancreatitis animal models is described. Significant analgesia and protection of pancreatic tissue was provided for the duration of the transgene expression (approximately 4-6 weeks). These studies establish a basis for use of HSV-based gene therapy for chronic visceral pain. Targeted enkephalin gene therapy approaches are providing clear promise for pain control. As innovative means of significantly reducing pancreatic inflammation and preserving tissue architecture, they may extend their clinical usefulness for pancreatitis and pancreatic cancer pain patients.

Plasticity in intact A delta- and C-fibers contributes to cold hypersensitivity in neuropathic rats.

Cold hypersensitivity is a common sensory abnormality accompanying peripheral neuropathies and is difficult to treat. Progress has been made in understanding peripheral mechanisms underlying neuropathic pain but little is known concerning peripheral mechanisms of cold hypersensitivity. The aim of this study was to analyze the contribution of uninjured primary afferents to the cold hypersensitivity that develops in neuropathic rats. Rats with a lumbar 5 (L5) and L6 spinal nerve ligation (SNL, Chung model) but not sham, developed mechanical allodynia, evidenced by decreased paw withdrawal thresholds and increased magnitude of response to von Frey stimulation. Cold hypersensitivity also developed in SNL but not sham rats, evidenced by enhanced nociceptive behaviors induced by placement on a cold plate (6 degrees C) or application of icilin (a transient receptor potential M8 (TRPM8)/transient receptor potential A1 (TRPA1) receptor agonist) to nerve-injured hind paws. Single fiber recordings demonstrated that the mean conduction velocities of intact L4 cutaneous A delta- and C-fibers were not different between naive and SNL rats; however, mechanical thresholds of the A delta- but not the C-fibers were significantly decreased in SNL compared with naive. There was a higher prevalence of C-mechanoheat-cold (CMHC) fibers in SNL compared with naive, but the overall percentage of cold-sensitive C-fibers was not significantly increased compared with naive. This was in contrast to the numerous changes in A delta-fibers: the percentage of L4 cold sensitive A delta-, but not C-fibers, was significantly increased, the percentage of L4 icilin-sensitive A delta-, but not C-fibers, was significantly increased, the icilin-induced activity of L4 A delta-, but not C-fibers, was significantly increased. Icilin-induced activity was blocked by the TRPA1 antagonist Ruthenium Red. The results indicate plasticity in both A delta- and C-uninjured fibers, but A delta fibers appear to provide a major contribution to cold hypersensitivity in neuropathic rats.

Upregulation of the phosphorylated form of CREB in spinothalamic tract cells following spinal cord injury: relation to central neuropathic pain.

Spinal cord injury (SCI) often leads to the generation of chronic intractable neuropathic pain. The mechanisms that lead to chronic central neuropathic pain (CNP) following SCI are not well understood, resulting in ineffective treatments for pain relief. Studies have demonstrated persistent hyperexcitability of dorsal horn neurons which may provide a substrate for CNP. We propose a number of similarities between CNP mechanisms and mechanisms that occur in long-term potentiation, in which hippocampal neurons are hyperexcitable. One biochemical similarity may be activation of the transcription factor, cyclic AMP response element-binding protein (CREB), via phosphorylation (pCREB). The current study was designed to examine whether tactile allodynia that develops in segments rostral to SCI (at-level pain) correlates with an increase in CREB phosphorylation in specific neurons known to be involved in allodynia, the spinothalamic tract (STT) cells. This study determined that, in animals experiencing at-level allodynia 35 days after SCI, pCREB was upregulated in the spinal cord segment rostral to the injury. In addition, pCREB was found to be upregulated specifically in STT cells in the rostral segment 35 days after SCI. These findings suggest one mechanism of maintained central neuropathic pain following SCI involves persistent upregulation of pCREB expression within STT cells.

Exogenous Bcl-xL fusion protein spares neurons after spinal cord injury.

Spinal cord injury (SCI) induces neuronal death, including apoptosis, which is completed within 24 hr at and around the impact site. We identified early proapoptotic transcriptional changes, including upregulation of proapoptotic Bax and downregulation of antiapoptotic Bcl-xL, Bcl-2, and Bcl-w, using Affymetrix DNA microarrays. Because Bcl-xL is the most robustly expressed antiapoptotic Bcl-2 molecule in adult central nervous system, we decided to characterize better the effect of SCI on Bcl-xL expression. We found Bcl-xL expressed robustly throughout uninjured spinal cord in both neurons and glia cells. We also found Bcl-xL localized in different cellular compartments: cytoplasmic, mitochondrial, and nuclear. Bcl-xL protein levels decreased in the cytoplasm and mitochondria 2 hr after SCI and persisted for 24 hr. To test the contribution of proapoptotic decreases in Bcl-xL to neuronal death, we augmented endogenous Bcl-xL levels by administering Bcl-xL fusion protein (Bcl-xL FP) into injured spinal cords. Bcl-xL FP significantly increased neuronal survival, suggesting that SCI-induced changes in Bcl-xL contribute considerably to neuronal death. Because Bcl-xL FP increases survival of dorsal horn neurons and ventral horn motoneurons, it could become clinically relevant in preserving sensory and motor functions after SCI.

Excitatory amino acids, TNF-alpha, and chemokine levels in synovial fluids of patients with active arthropathies.

The aim of this study was to assess the synovial fluid (SF) neurotransmitter excitatory amino acid (EAA) levels, including glutamate (Glu) and aspartate (Asp), in the context of SF levels of other amino acids, TNF-alpha and chemokines from patients with active arthropathies. The SF was collected from patients with active rheumatoid arthritis (RA), gout, or osteoarthritis (OA). The SF samples were analysed for levels of neurotransmitters glutamate and aspartate, tumour necrosis factor-alpha (TNF-alpha), Regulated upon Activation Normally T-cell Expressed and Secreted (RANTES), macrophage inhibitory factor-1 alpha (MIP-1alpha) and interleukin 8 (IL-8). SF WBC counts were also determined. Correlations between SF EAA, TNF-alpha and chemokines were determined by the Pearson product-moment correlation. Primary cultures derived from SF from active RA and gout patients were incubated with added l-glutamate, to assess if exposure to Glu could increase TNF-alpha levels. There were significant elevations in SF EAA, SF TNF-alpha and SF RANTES in RA patients compared to gout or OA patients. Significant correlations between SF EAA and SF RANTES, MIP-1alpha and IL-8 levels were seen, and SF EAA and SF TNF-alpha or SF WBC levels approached significance. Addition of exogenous neurotransmitter glutamate significantly increased TNF-alpha levels in primary cell cultures derived from RA and gout patients. The SF neurotransmitter EAA levels significantly correlated to selected SF chemokine levels, in clinically active RA, gout and OA patients, independent of disease. Added Glu resulted in significantly increased TNF-alpha levels in primary synovial cell cultures. These data expand the relationship of SF neurotransmitter EAA levels to SF cytokines and chemokines in patients with clinically active arthritis, and suggest that neurotransmitters Glu and Asp contribute to peripheral inflammatory processes.

Comparison of microdialysis and push-pull perfusion for retrieval of serotonin and norepinephrine in the spinal cord dorsal horn.

Both push-pull and microdialysis methods are utilized to measure norepinephrine and serotonin in the dorsal horn of the spinal cord. This experiment was designed to determine which technique is better for measurement of norepinephrine and serotonin in the spinal cord and also to determine if the samples are best collected with or without perchloric acid. Sample stability and an assay validation for precision, limit of quantification, and limit of detection were also performed. Push-pull or microdialysis catheters were placed transversely through the dorsal horn and the catheter was perfused with artificial cerebrospinal fluid. Noxious pinch (20 s/min for 10 min) was used to evoke a change in the concentration of catecholamines. Samples were collected before, during and after pinch. No basal concentrations of epinephrine and serotonin were found with microdialysis. Although basal concentrations of norepinephrine were measured by microdialysis, there was no change in response to noxious pinch. The push-pull technique coupled with collection of samples without perchloric acid showed that significant increases in serotonin and norepinephrine are measurable in response to noxious pinch. In contrast, when samples were collected with perchloric acid present there was no change in serotonin or norepinephrine in response to pinch. The stability of catecholamines is greatly affected by perchloric acid such that there is a near complete loss of ability to detect serotonin and norepinephrine by 24 h in samples collected by push-pull. In contrast, samples collected without perchloric acid showed only a 20% reduction in concentration by 24 h. Even without perchloric acid, by 1 wk there was a 50% or greater loss in the concentrations of norepinephrine in push-pull samples. Thus, to measure changes in catecholamines in the dorsal horn, push-pull collected without perchloric provides measurable, reliable and valid results if analyzed by high performance liquid chromatography within 24 h.

DNA microarray analysis of the contused spinal cord: effect of NMDA receptor inhibition.

Spinal cord injury (SCI)-induced neurodegeneration leads to irreversible and devastating motor and sensory dysfunction. Post-traumatic outcomes are determined by events occurring during the first 24 hours after SCI. An increase in extracellular glutamate concentration to neurotoxic levels is one of the earliest events after SCI. We used Affymetrix DNA oligonucleotide microarrays (with 1,322 DNA probes) analysis to measure gene expression in order to test the hypothesis that SCI-induced N-methyl-D-aspartate (NMDA) receptor activation triggers significant postinjury transcriptional changes. Here we report that SCI, 1 hour after trauma, induced change in mRNA levels of 165 genes and expression sequence tags (ESTs). SCI affected mRNA levels of those genes that regulate predominantly transcription factors, inflammation, cell survival, and membrane excitability. We also report that NMDA receptor inhibition (with -(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine hydrogen maleate [MK-801]) reversed the effect of SCI on about 50% of the SCI-affected mRNAs. Especially interesting is the finding that NMDA receptor activation participates in the up-regulation of inflammatory factors. Therefore, SCI-induced NMDA receptor activation is one of the dominant, early signals after trauma that leads to changes in mRNA levels of a number of genes relevant to recovery processes. The majority of MK-801 effects on the SCI-induced mRNA changes reported here are novel. Additionally, we found that the MK-801 treatment also changed the mRNA levels of 168 genes and ESTs that had not been affected by SCI alone, and that some of their gene products could have harmful effects on SCI outcome.