[Show abstract][Hide abstract] ABSTRACT: Agrin, a heparan sulfate proteoglycan functioning as a neuro-muscular junction inducer, has been shown to inhibit neuropathic pain in sciatic nerve injury rat models, via phosphorylation of N-Methyl-D-aspartate receptor NR1 subunits in gamma-aminobutyric acid neurons. However, its effects on spinal cord injury-induced neuropathic pain, a debilitating syndrome frequently encountered after various spine trauma, are unknown. In the present investigation, we studied the 50 kDa agrin isoform effects in a quisqualic acid dorsal horn injection rat model mimicking spinal cord injury-induced neuropathic pain. Our results indicate that 50 kDa agrin decreased only in the dorsal horn of neuropathic animals and increased 50 kDa agrin expression in the dorsal horn, via intra-spinal injection of adeno-associated virus serum type two, suppressed spinal cord injury-induced neuropathic pain. Also, the reason why 50 kDa agrin only activates the N-Methyl-D-aspartate receptor NR1 subunits in the GABA neurons, but not in sensory neurons, is unknown. Using immunoprecipitation and western-blot analysis, two dimensional gel separation, and mass spectrometry, we identified several specific proteins in the reaction protein complex, such as neurofilament 200 and mitofusin 2, that are required for the activation of the NR1 subunits of gamma-aminobutyric acid inhibitory neurons by 50 kDa agrin. These findings indicate that 50 kDa agrin is a promising agent for neuropathic pain treatment.
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND:: Resiniferatoxin (RTX), an excitotoxic agonist for vanilloid receptor 1, is a promising candidate for intractable pain treatment. OBJECTIVE:: We evaluated the effects of RTX, applied to dorsal root ganglia (DRG) at high doses (1200 ng), in sensory-motor function and nerve growth factor (NGF) alterations in a photochemical sciatic nerve injury rat model. METHODS:: Following RTX injection into the L3-6 DRG at high doses and behavioral evaluation, the rats were sacrificed and the DRG were tested by immunohistochemistry and mRNA analysis for NGF and its' receptors, tyrosine kinase A (TrkA) and p75. The correlation between neuropathic pain and NGF, TrkA, and p75 expression was analyzed. RESULTS:: The treated rats had preserved touch, cold, pain, and high-heat sensations, and exhibited hypoalgesia to low-heat stimulation. After RTX treatment, TrkA and p75 altered their expressions from one neuronal type to another in the DRG. NGF and p75 expression changed from the small-size neurons in neuropathic rat DRG to the large- and medium-size neurons in non-neuropathic and RTX-treated animals, concomitantly with neuropathic pain suppression. TrkA was expressed in the small-size neurons in neuropathic rat DRG, and was drastically reduced in all size neurons after RTX treatment. NGF, TrkA, and p75 mRNA expression supported these phenotypic changes in the DRG. CONCLUSION:: The pathway of NGF-TrkA expressed in the small-size neurons, associated with neuropathic pain, was shifted to the NGF-p75 pathway expressed in the large-size neurons after RTX treatment, in association with neuropathic pain inhibition. These findings may play an important role in clinical trial designs.
[Show abstract][Hide abstract] ABSTRACT: Micro RNAs (miRNAs) constitute a unique class of small, non-coding ribonucleic acids (RNAs) that regulate gene expression at the post-transcriptional level. The presence of two inducible miRNAs, miRNA-125b and miRNA-146a, involved in respectively, astroglial cell proliferation and in the innate immune and inflammatory response, is significantly up-regulated in human neurological disorders including Alzheimer's disease (AD). In this study we analyzed abundances miRNA-125b and miRNA-146a in magnesium-, iron-, gallium, and aluminum-sulfate-stressed human-astroglial (HAG) cells, a structural and immune-responsive brain cell type. The combination of iron- plus aluminum-sulfate was found to be significantly synergistic in up-regulating reactive oxygen species (ROS) abundance, NF-кB-DNA binding and miRNA-125b and miRNA-146a expression. Treatment of metal-sulfate stressed HAG cells with the antioxidant phenyl butyl nitrone (PBN) or the NF-кB inhibitors curcumin, the metal chelator-anti-oxidant pyrollidine dithiocarbamate (PDTC), or the resveratrol analog CAY10512, abrogated both NF-кB signaling and induction of these miRNAs. Our observations further illustrate the potential of physiologically relevant amounts of aluminum and iron sulfates to synergistically up-regulate specific miRNAs known to contribute to AD-relevant pathogenetic mechanisms, and suggest that antioxidants or NF-кB inhibitors may be useful to quench metal-sulfate triggered genotoxicity.
[Show abstract][Hide abstract] ABSTRACT: Neurotrophin-3 (NT3) and its cognate receptor, tyrosine kinase C (TrkC), have recently been shown to modulate neuropathic pain. Another receptor, the transient receptor potential vanilloid 1, is considered a molecular integrator for nociception. Transient receptor potential vanilloid 1-positive cells can be selectively ablated by Resiniferatoxin (RTX). NT3 changes in the dorsal root ganglia (DRG) after RTX treatment may further define their role in pain modulation.
To demonstrate the role of NT3 and TrkC in intraganglial RTX-induced pain suppression and in neuropathic pain development.
Fifty-three rats underwent a photochemical left sciatic nerve injury. Neuropathic animals were treated by RTX injection in the ipsilateral L3-6 DRG. NT3 and TrkC presence in the DRG was evaluated before and after the nerve injury, as well as after RTX treatment.
The RTX injection resulted in pain inhibition. NT3 normally expressed mainly in large- and medium-size neurons. NT3 presence was increased mainly in the small DRG cells of neuropathic animals, and the medium- and large-size neurons of nonallodynic rats. RTX treatment of allodynic rats changed the NT3 distribution to a nonallodynic pattern. TrkC expressed mainly in large/medium-size neurons. After nerve injury, TrkC expression was also increased in the small DRG cells of allodynic animals (although less than NT3), and the medium- and large-size cells of nonallodynic ones. After RTX, TrkC expression gradually decreased, but with persistence in the large DRG cells.
NT3 may have antinociceptive effects in the DRG. These effects may be mediated, at least in part, by TrkC in the medium- and large-size DRG neurons.
[Show abstract][Hide abstract] ABSTRACT: The elusiveness of neuropathic pain mechanisms is a major impediment in developing effective clinical treatments. Here we show that peripheral nerve injury decreased agrin expression in the ipsilateral spinal dorsal horn of rats displaying tactile allodynia. SCP1, an acetaminophen analog, suppressed allodynia and promoted agrin upregulation. Preemptive treatment with SCP1 also upregulated agrin, thereby preventing neuropathic pain development. Expression of 50 kDa agrin delivered by adeno-associated virus into the dorsal horn also suppressed allodynia and hyperalgesia. Allodynia suppression was a consequence of serine residue 896/897 phosphorylation of NMDA receptor NR1 subunits in the GABA interneurons of the dorsal horn. Agrin silencing by small interference RNA, administered with either AAV-Ag50 vector or SCP1, blocked allodynia suppression, agrin upregulation, and NR1 phosphorylation. In conclusion, 50 kDa agrin modulates neuropathic pain through NR1 phosphorylation in GABA neurons. This mechanism may open new approaches for treating not only neuropathic pain, but also epilepsy, tremors, and spasticity.
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 11/2010; 30(45):15286-97. DOI:10.1523/JNEUROSCI.4418-10.2010 · 6.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Brain-derived neurotrophic factor (BDNF) and its cognate receptor, the tyrosine kinase B (TrkB), are normally expressed in neurons and implicated in multiple pathological conditions. Brain-derived neurotrophic factor is produced in the central nervous system microglia in response to noxious stimuli and appear to potentiate central sensitization. Resiniferatoxin (RTX) is an excitotoxic agonist of the vanilloid receptor 1 (VR1), a cation channel protein considered an integrator for nociception. Resiniferatoxin, administered into the dorsal root ganglia (DRG), selectively eliminates the VR1-positive neurons and improves tactile allodynia in a neuropathic pain rat model.
The goal of the present study was to evaluate the role of BDNF in RTX-induced neuropathic pain suppression.
The study design was a sciatic nerve injury animal model with intraganglionic RTX injection.
Resiniferatoxin was injected into the DRG of the L3-L6 spinal nerves after the rats displayed tactile allodynia and thermal hyperalgesia produced by a photochemical injury to the sciatic nerve. Behavioral testing and immunohistochemical and mRNA analysis of the DRG were performed to determine BDNF's role in pain modulation.
Brain-derived neurotrophic factor expression in the DRG of neuropathic rats was upregulated in the small- and medium-size neurons, whereas the upregulation was observed in the large-size neurons of non-neuropathic rat DRG. A high-dose RTX injection in the DRG of neuropathic rats led to elimination of both thermal hyperalgesia and tactile allodynia and also upregulated BDNF in the large-size neurons, similar to the nonallodynic rats. Tyrosine kinase B changes mirrored the BDNF ones.
Resiniferatoxin injection in the DRG of neuropathic rats upregulates BDNF expression in the same pattern as in the large-size neurons of non-neuropathic rats. Therefore, BDNF upregulation may have pain suppressive effects. These effects are likely mediated by TrkB.
The spine journal: official journal of the North American Spine Society 05/2010; 10(8):715-20. DOI:10.1016/j.spinee.2010.03.029 · 2.43 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Micro RNAs (miRNAs) represent a family of small ribonucleic acids (RNAs) that are post-transcriptional regulators of messenger RNA (mRNA) complexity. Brain cells maintain distinct populations of miRNAs that support physiologically normal patterns of expression, however, certain miRNA abundances are significantly altered in neurodegenerative disorders such as Alzheimer's disease (AD). Here we provide evidence in human neural (HN) cells of an aluminum-sulfate- and reactive oxygen species (ROS)-mediated up-regulation of an NF-kappaB-sensitive miRNA-146a that down-regulates the expression of complement factor H (CFH), an important repressor of inflammation. This NF-kappaB-miRNA-146a-CFH signaling circuit is known to be similarly affected by Abeta42 peptides and in AD brain. These aluminum-sulfate-inducible events were not observed in parallel experiments using iron-, magnesium-, or zinc-sulfate-stressed HN cells. An NF-kappaB-containing miRNA-146a-promoter-luciferase reporter construct transfected into HN cells showed significant up-regulation of miRNA-146a after aluminum-sulfate treatment that corresponded to decreased CFH gene expression. These data suggest that (1) as in AD brain, NF-kappaB-sensitive, miRNA-146a-mediated, modulation of CFH gene expression may contribute to inflammatory responses in aluminum-stressed HN cells, and (2) underscores the potential of nanomolar aluminum to drive genotoxic mechanisms characteristic of neurodegenerative disease processes.
[Show abstract][Hide abstract] ABSTRACT: Glioma and glioblastoma multiforme constitute rapidly proliferating glial cell tumors whose pathogenic mechanisms are not well understood. This study examined proinflammatory and neurodegenerative gene expression in five American Tissue Culture Collection glioma and glioblastoma multiforme tumor cell lines and in 14 glioma and glioblastoma samples obtained from human brain biopsy. Expression of the low-abundance cyclooxygenase-1 and the high-abundance cytoskeletal element beta-actin were found not to significantly change in any cells or tissues studied and were used as internal controls. In contrast, proinflammatory cyclooxygenase-2, cytosolic phospholipase A2, IL-1beta, and beta-amyloid precursor protein expression levels were found to be significantly upregulated. These studies suggest that glioma and glioblastoma exhibit robust upregulation of proinflammatory and neurodegenerative genetic markers that may contribute to the pathobiology, phenotype, and proliferation of glial cell growth.
[Show abstract][Hide abstract] ABSTRACT: The vanilloid receptor 1 (VR1) is expressed by the type II A-delta and C-fiber neurons, functioning as a molecular integrator for nociception. VR1 can be selectively ablated by resiniferatoxin (RTX), an ultra-potent excitotoxic agonist, when injected into sensory ganglia.
To evaluate the role of the VR1-positive neurons in neuropathic pain.
Photochemical injury to rat sciatic nerve (Gazelius model).
Two groups of rats underwent the photochemical injury and RTX treatment. RTX was injected in the dorsal root ganglia (DRGs) of the L3, L4, L5, and L6 nerve roots, either after or before the nerve injury. The animals were tested for thermal hyperalgesia (noxious heat stimuli) and mechanical allodynia (von Frey filaments). Immunohistochemical analysis of the DRGs was performed after euthanasia.
In the tactile allodynic rats, RTX injection in the DRGs improved the average withdrawal threshold from 1.62 g to 5.68 g. Immunohistochemical labeling showed that almost all VR1-positive neurons were eliminated. When RTX was administrated into the ipsilateral DRGs before the nerve injury, this treatment prevented the development of tactile allodynia in 12 out of 14 rats. Immunohistochemical staining revealed that the VR1-positive neurons were eliminated in the rats that did not develop tactile allodynia, whereas they were still present in the allodynic rats.
VR1-positive neurons are essential for the development of mechanical allodynia. In rats already exhibiting neuropathic pain, the VR1-positive neurons mediate the most sensitive part of mechanical allodynia. RTX injection in sensory ganglia may represent a novel treatment for neuropathic pain.
The Spine Journal 03/2008; 8(2):351-8. DOI:10.1016/j.spinee.2007.08.005 · 2.43 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Platelet activating factor (PAF; beta-acetyl-gamma-O-hexadecyl-l-alpha-phosphatidylcholine) triggers a rapid pro-inflammatory gene expression program in primary cultures of human neural (HN) cells. Two genes and gene products consistently induced after PAF treatment are the cytosoluble prostaglandin synthase cycloooxygenase-2 (COX-2) and the pro-apoptotic tumor necrosis factor alpha (TNFalpha). Both of these mediators are associated with the activation of inflammatory signaling, neural cell dysfunction, apoptosis and brain cell death, and both have been found to be up-regulated after brain injury in vivo. In this study we investigated the effects of the non-halogenated synthetic glucocorticoid budesonide epimer R (BUDeR), the novel PAF antagonist LAU-0901, and the electron spin trap and free radical scavenger phenyl butyl nitrone (PBN), upon early COX-2 and TNFalpha gene activation and prostaglandin E(2) (PGE(2)) release in PAF-stressed primary HN cells. The data indicate that these three biochemically unrelated classes of inflammatory repressors act synergistically in modulating PAF-induced up-regulation of COX-2, TNFalpha, and PGE(2) by quenching oxidative stress or inflammatory signaling, resulting in increased HN cell survival. These, or analogous classes of compounds, may be useful in the design of more effective combinatorial pharmacotherapeutic strategies in the treatment of complex neuro-inflammatory disorders.
[Show abstract][Hide abstract] ABSTRACT: Sortilin 1 (SORL1) is a transmembrane sorting receptor that regulates the intracellular trafficking of beta-amyloid precursor protein (betaAPP). Interactions between SORL1 and betaAPP result in the decreased processing of betaAPP into toxic amyloid-beta42 (Abeta42) peptides that accumulate in Alzheimer's disease brain. Here, we report selectively decreased levels of SORL1 in limbic and occipital regions of Alzheimer brain that inversely correlate with amyloid plaque and neurofibrillary tangle density. Reduced SORL1, coupled to elevated beta-amyloid cleaving enzyme, presenilin-1 and increased Abeta42 peptide secretion, was observed after incubation of cultured human neural cells with the proinflammatory cytokine interleukin-1beta. The results suggest that SORL1 deficits may not only promote the pathogenic processing of betaAPP but may also contribute to Abeta42-mediated inflammatory signaling in stressed human brain cells.
[Show abstract][Hide abstract] ABSTRACT: Alzheimer's disease is associated with progressively dysfunctional gene expression in the limbic system of the brain. The thalamus and primary visual cortex are thought to be initially spared of Alzheimer-type changes that ravage the association neocortex. In this study, using DNA arrays and Western immunoassay, gene expression patterns were examined in the thalamus and primary visual cortex of moderate-stage and late-stage Alzheimer's disease and age-matched controls using a set of proinflammatory genes known to be upregulated in the temporal lobe neocortex and hippocampus of moderate-stage Alzheimer's disease. The data indicate that, in late-stage Alzheimer's disease, proinflammatory and proapoptotic gene expression spreads into the primary visual sensory cortex. This upregulation of pathological gene expression could be, in part, responsible for the visual disturbances associated with end-stages of the Alzheimer process.
[Show abstract][Hide abstract] ABSTRACT: Neurons, glia, and endothelial cells of the cerebral microvasculature co-exist in intimate proximity in nervous tissues, and their homeostatic interactions in health, as well as coordinated response to injury, have led to the concept that they form the basic elements of a functional neurovascular unit. During the course of normal cellular metabolism, growth, and development, each of these brain cell types secrete various species of potentially neurotoxic peptides and factors, events that increase in magnitude as brain cells age. This article reviews contemporary research on the secretory products of the three primary cell types that constitute the neurovascular unit in deep brain regions. We provide some novel in vitro data that illustrate potentially pathogenic paracrine effects within primary cells of the neurovascular unit. For example, the pro-inflammatory cytokine interleukin (IL)-1beta was found to stimulate amyloid-beta (Abeta) peptide release from human neural cells, and human brain microvessel endothelial cells exposed to transient hypoxia were found to secrete IL-1beta at concentrations known to induce Abeta42 peptide release from human neural cells. Hypoxia and excessive IL-1beta and Abeta42 abundance are typical pathogenic stress factors implicated in the initiation and development of common, chronic neurological disorders such as Alzheimer's disease. These data support the hypothesis that paracrine effects of stressed constituent cells of the neurovascular unit may contribute to "spreading effects" characteristic of progressive neurodegenerative disorders.
[Show abstract][Hide abstract] ABSTRACT: SCP-1, n-[alpha-(benzisothiazol-3(2ho-ona,1-dioxide-2yl)-acetyl]-p-aminophenol (100 nmol), when intrathecally injected, suppressed tactile allodynia and thermal hyperalgesia in a rat neuropathic pain model. The tactile allodynia suppression lasted for at least 4h and SCP-M1 (100 nmol), the main metabolite of SCP-1, displayed similar suppression as SCP-1, but shorter latency, indicating SCP-M1 may be the bioactive component of SCP-1. Acetaminophen was less potent than SCP-1 and SCP-M1. To study mechanisms underlying SCP-1 action, we recorded voltage-gated Ca(2+) channel currents in acutely isolated dorsal root ganglion neurons using the whole-cell patch-clamp technique. SCP-1 and SCP-M1 inhibited non-L-type calcium channel currents up to 23.0+/-2.3% and 23.1+/-3.5%, respectively, at a depolarized pulse to -10 mV from a holding potential of -80 mV. Acetaminophen only induced 6.8+/-1.0% inhibition. The results suggest SCP-1 possesses anti-nociceptive activity in the rat model involving calcium channel blocking properties.
Biochemical and Biophysical Research Communications 12/2006; 350(2):358-63. DOI:10.1016/j.bbrc.2006.09.055 · 2.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To investigate the expression of cyclooxygenase (COX)-1, -2, and -3 RNA and protein in retinal pigment epithelial (ARPE-19) cells and in human neural (HN) cells exposed to the stress-inducing cytokines IL-1beta and TNF-a, the oxidizing peroxide H(2)O(2), the combination of TNF-alpha + H(2)O(2), and the lipofuscin fluorophore A2E.
Three-week-old ARPE-19 and HN cells were incubated with IL-1beta (10 ng/ml), TNF-alpha (10 ng/ml), H(2)O(2) (0.6 microM), TNF-alpha + H(2)O(2) (10 ng/ml and 0.6 microM), or A2E (10 microM) for 8 hr, after which total RNA and whole cellular proteins were isolated. Cyclooxygenase-1, -2, and -3 RNA and protein levels were quantified using Northern and Western immunoassay.
IL-1beta-, H(2)O(2)-, TNF-alpha-, TNF-alpha + H(2)O(2)-, or A2E-stressed ARPE-19 or HN cells displayed no significant upregulation in COX-1 or COX-3 RNA message abundance; however, significant upregulation was observed in COX-2 RNA message and protein abundance. A2E treatment of HN cells resulted in modest increases in COX-3 protein, an effect that was not observed in ARPE-19 cells.
COX-2 RNA levels were induced in cytokine-, peroxide-, and A2E-stressed ARPE-19 and HN cells. Lack of induction of COX-3 RNA message by A2E, coupled with increases in COX-3 protein under identical treatment conditions, suggest that significant post-transcriptional or post-translational controls may regulate COX-3 gene expression in HN cells. Stress-induced upregulation of COX-2 gene expression in ARPE-19 and HN cells may play a mechanistic role in promoting proinflammatory and/or pro-oxidative pathology in these tissues.
Current Eye Research 04/2006; 31(3):259-63. DOI:10.1080/02713680600556974 · 1.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Transcription from the human cytosolic phospholipase A2 gene has been observed to be hypoxia sensitive in endothelial cells cultured from the human cerebral microvasculature. DNA sequence analysis of the cytosolic phospholipase A2 promoter revealed the presence of a distal cluster of potential hypoxia-inducible factor-1-DNA binding sites homologous to 5'-NCGTG-3', located between -1087 and -996 bp of the major start of transcription at +1 bp (Genbank U08374). Gel shift assay showed strong hypoxia-inducible factor-1-DNA binding to only a single site within this cluster, and promoter deletion analysis indicated the functional importance of this chromatin domain in conveying oxygen sensitivity to cytosolic phospholipase A2 gene transcription. Non-functional hypoxia inducible factor-1-DNA binding sites flanking a single functional hypoxia-inducible factor-1-DNA binding site in this hypoxia-sensitive domain may promote oxygen sensitivity via transcription factor clustering or Circe effects.