Michael A. Henry

Glial cell line-derived neurotrophic factor augments midbrain dopaminergic circuits in vivo

Recently, a novel glial cell line-derived neurotrophic factor (GDNF) has been identified, cloned, and shown to have potent survival- and growth-promoting activity on fetal rat midbrain dopaminergic neurons in cell culture. In this study, we document marked and long-lasting effects on adult rat midbrain dopaminergic neurons in vivo after intracranial administration. A single injection of this factor into the substantia nigra elicited a dose-dependent increase in both spontaneous and amphetamine-induced motor activity, and a decrease in food consumption, lasting 7-10 days. Using immunocytochemistry, we found sprouting of tyrosine hydroxylase-positive neurites towards the injection site, and increased tyrosine hydroxylase immunoreactivity of the ipsilateral striatum was produced by GDNF. There was also a marked and dose-dependent increase in dopamine turnover in the substantia nigra and striatum, and in ipsilateral dopamine levels in the substantia nigra. Little or no effects of GDNF were seen on norepinephrine or serotonin levels. The neurochemical changes on dopaminergic afferents persist for at least 3 weeks after a single intracranial injection of 10 micrograms. Taken together, these data suggest that this glial cell line-derived factor has a potent influence on adult rat dopamine neurons and may have a potentially important role as a trophic factor for these neurons.

Evaluation of the Delivery of Mesenchymal Stem Cells into the Root Canal Space of Necrotic Immature Teeth after Clinical Regenerative Endodontic Procedure

INTRODUCTION Immature teeth with open apices treated with conventional nonsurgical root canal treatment often have a poor prognosis as a result of the increased risk of fracture and susceptibility to recontamination. Regenerative endodontics represents a new treatment modality that focuses on reestablishment of pulp vitality and continued root development. This clinical procedure relies on the intracanal delivery of a blood clot (scaffold), growth factors (possibly from platelets and dentin), and stem cells. However, to date, the clinical presence of stem cells in the canal space after this procedure has not been demonstrated. The purpose of this clinical study was to evaluate whether regenerative endodontic procedures are able to deliver stem cells into the canal space of immature teeth in young patients and to identify the possible tissue origin for these cells. METHODS After informed consent, the first appointment consisted of NaOCl irrigation and treatment with a triple antibiotic paste. One month later, the root canal space was irrigated with sterile saline, and bleeding was evoked with collection of samples on paper points. Real-time reverse-transcription polymerase chain reaction and immunocytochemistry were conducted to compare the gene transcripts and proteins found in the root canal sample with levels found in the systemic circulation. RESULTS Molecular analyses of blood collected from the canal system indicated the significant accumulation of transcripts for the stem cell markers CD73 and CD105 (up to 600-fold), compared with levels found in the systemic blood. Furthermore, this effect was selective because there was no change in expression of the differentiation markers ALK-P, DSPP, ZBTB16, and CD14. Histologic analyses demonstrated that the delivered cells expressed both CD105 and STRO-1, markers for a subpopulation of mesenchymal stem cells. CONCLUSIONS Collectively, these findings demonstrate that the evoked-bleeding step in regenerative procedures triggers the significant accumulation of undifferentiated stem cells into the canal space where these cells might contribute to the regeneration of pulpal tissues seen after antibiotic paste therapy of the immature tooth with pulpal necrosis.

LPS Sensitizes TRPV1 via Activation of TLR4 in Trigeminal Sensory Neurons

Recent studies have demonstrated that the lipopolysaccharide (LPS) receptor (TLR4) is expressed in TRPV1 containing trigeminal sensory neurons. In this study, we evaluated whether LPS activates trigeminal neurons, and sensitizes TRPV1 responses via TLR4. To test this novel hypothesis, we first demonstrated that LPS binds to receptors in trigeminal neurons using competitive binding. Second, we demonstrated that LPS evoked aconcentration-dependent increase in intracellular calcium accumulation (Ca2+)i and inward currents. Third, LPS significantly sensitized TRPV1 to capsaicin measured by (Ca2+)i, release of calcitonin gene-related peptide, and inward currents. Importantly, a selective TLR4 antagonist blocked these effects. Analysis of these data, collectively, demonstrates that LPS is capable of directly activating trigeminal neurons, and sensitizing TRPV1 via a TLR4-mediated mechanism. These findings are consistent with the hypothesis that trigeminal neurons are capable of detecting pathogenic bacterial components leading to sensitization of TRPV1, possibly contributing to the inflammatory pain often observed in bacterial infections.

Sodium channel Nav1.6 accumulates at the site of infraorbital nerve injury

BackgroundSodium channel (NaCh) expressions change following nerve and inflammatory lesions and this change may contribute to the activation of pain pathways. In a previous study we found a dramatic increase in the size and density of NaCh accumulations, and a remodeling of NaChs at intact and altered myelinated sites at a location just proximal to a combined partial axotomy and chromic suture lesion of the rat infraorbital nerve (ION) with the use of an antibody that identifies all NaCh isoforms. Here we evaluate the contribution of the major nodal NaCh isoform, Nav1.6, to this remodeling of NaChs following the same lesion. Sections of the ION from normal and ION lesioned subjects were double-stained with antibodies against Nav1.6 and caspr (contactin-associated protein; a paranodal protein to identify nodes of Ranvier) and then z-series of optically sectioned images were captured with a confocal microscope. ImageJ (NIH) software was used to quantify the average size and density of Nav1.6 accumulations, while additional single fiber analyses measured the axial length of the nodal gap, and the immunofluorescence intensity of Nav1.6 in nodes and of caspr in the paranodal region.ResultsThe findings showed a significant increase in the average size and density of Nav1.6 accumulations in lesioned IONs when compared to normal IONs. The results of the single fiber analyses in caspr-identified typical nodes showed an increased axial length of the nodal gap, an increased immunofluorescence intensity of nodal Nav1.6 and a decreased immunofluorescence intensity of paranodal caspr in lesioned IONs when compared to normal IONs. In the lesioned IONs, Nav1.6 accumulations were also seen in association with altered caspr-relationships, such as heminodes.ConclusionThe results of the present study identify Nav1.6 as one isoform involved in the augmentation and remodeling of NaChs at nodal sites following a combined partial axotomy and chromic suture ION lesion. The augmentation of Nav1.6 may result from an alteration in axon-Schwann cell signaling mechanisms as suggested by changes in caspr expression. The changes identified in this study suggest that the participation of Nav1.6 should be considered when examining changes in the excitability of myelinated axons in neuropathic pain models.

Concentration-dependent effect of sodium hypochlorite on stem cells of apical papilla survival and differentiation.

INTRODUCTION Intracanal disinfection is a crucial step in regenerative endodontic procedures. Most published cases suggest the use of sodium hypochlorite (NaOCl) as the primary irrigant. However, the effect of clinically used concentrations of NaOCl on the survival and differentiation of stem cells is largely unknown. In this study, we tested the effect of various concentrations of NaOCl on the stem cells of the apical papilla (SCAPs) survival and dentin sialophosphoprotein (DSPP) expression. METHODS Standardized root canals were created in extracted human teeth and irrigated with NaOCl (0.5%, 1.5%, 3%, or 6%) followed by 17% EDTA or sterile saline. SCAPs in a hyaluronic acid-based scaffold were seeded into the canals and cultured for 7 days. Next, viable cells were quantified using a luminescence assay, and DSPP expression was evaluated using quantitative real-time polymerase chain reaction. RESULTS There was a significant reduction in survival and DSPP expression in the group treated with 6% NaOCl compared with the untreated control group. Comparable survival was observed in the groups treated with the lower concentrations of NaOCl, but greater DSPP expression was observed in the 1.5% NaOCl group. In addition, 17% EDTA resulted in increased survival and DSPP expression partially reversing the deleterious effects of NaOCl. CONCLUSIONS Collectively, the results suggest that dentin conditioning with high concentrations of NaOCl has a profound negative effect on the survival and differentiation of SCAPs. However, this effect can be prevented with the use of 1.5% NaOCl followed by 17% EDTA. The inclusion of this irrigation regimen might be beneficial in regenerative endodontic procedures.

6-Hydroxydopamine induces the loss of the dopaminergic phenotype in substantia nigra neurons of the rat

Intraparenchymal injections of the neurotoxin 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle in rats destroys the dopaminergic neurons in the pars compacta of the substantia nigra. In other transmitter systems it has been found that axotomy or neurotoxin exposure produces an initial loss of neurotransmitter phenotype, with cell death occurring over a much slower time course. To determine whether this also occurs in dopamine neurons after 6-OHDA, two approaches were utilized. First, the effect of injections of 6-OHDA into the medial forebrain bundle on nigral dopaminergic neurons was studied using combined fluorogold and immunocytochemical labeling. Four weeks after the 6-OHDA injection, there was an 85% reduction in the number of tyrosine hydroxylase (TH)-immunoreactive cells on the lesioned side. In contrast, there was only a 50% reduction in the number of fluorogold-labeled cells on the lesioned side. Second, the time course of the rescue of dopaminergic neurons after 6-OHDA by glial cell line-derived neurotrophic factor (GDNF) was determined using TH immunocytochemistry. Greater numbers of dopamine neurons were rescued 9 weeks after GDNF, compared with counts made 5 weeks after GDNF. Taken together, these results suggest loss of dopaminergic phenotype is greater than cell loss following 6-OHDA injections, and that GDNF restores the phenotype of affected cells.

A-Kinase Anchoring Protein Mediates TRPV1 Thermal Hyperalgesia through PKA Phosphorylation of TRPV1

&NA; Certain phosphorylation events are tightly controlled by scaffolding proteins such as A‐kinase anchoring protein (AKAP). On nociceptive terminals, phosphorylation of transient receptor potential channel type 1 (TRPV1) results in the sensitization to many different stimuli, contributing to the development of hyperalgesia. In this study, we investigated the functional involvement of AKAP150 in mediating sensitization of TRPV1, and found that AKAP150 is co‐expressed in trigeminal ganglia (TG) neurons from rat and associates with TRPV1. Furthermore, siRNA‐mediated knock‐down of AKAP150 expression led to a significant reduction in PKA phosphorylation of TRPV1 in cultured TG neurons. In CHO cells, the PKA RII binding site on AKAP was necessary for PKA enhancement of TRPV1‐mediated Ca2+‐accumulation. In addition, AKAP150 knock‐down in cultured TG neurons attenuated PKA sensitization of TRPV1 activity and in vivo administration of an AKAP antagonist significantly reduced prostaglandin E2 sensitization to thermal stimuli. These data suggest that AKAP150 functionally regulates PKA‐mediated phosphorylation/sensitization of the TRPV1 receptor.

A-kinase anchoring protein 150 controls protein kinase C-mediated phosphorylation and sensitization of TRPV1

ABSTRACT Post‐translational modifications on various receptor proteins have significant effects on receptor activation. For the Transient Receptor Potential family V type 1 (TRPV1) receptor, phosphorylation of certain serine/threonine amino acid residues sensitizes the receptor to activation by capsaicin and heat. Although Protein Kinase C (PKC) phosphorylates TRPV1 on certain serine/threonine residues, it is not completely understood how PKC functionally associates with TRPV1. Recent studies have reported that the A‐kinase Anchoring Protein 150 (AKAP150) mediates PKA phosphorylation of TRPV1 in several nociceptive models. Here, we demonstrate that AKAP150 also mediates PKC‐directed phosphorylation and sensitization of TRPV1. In cultured rat trigeminal ganglia, immunocytochemical analyses demonstrate co‐localization of AKAP150 and PKC isoforms α, δ, &egr;, and γ in TRPV1‐positive neurons. Additional biochemical evidence supports immunocytochemical results, indicating that AKAP150 preferentially associates with certain PKC isoforms in rat trigeminal ganglia neurons. Employing siRNA‐mediated knock‐down of AKAP150 expression, we demonstrate that PKC‐mediated phosphorylation of TRPV1 and sensitization to a capsaicin response is dependent upon functional expression of the AKAP150 scaffolding protein. Furthermore, PKC‐induced sensitization to a thermal stimulus is abrogated in AKAP150 knock‐out animals relative to wild‐type. Collectively, the results from these studies indicate that the AKAP150 scaffolding protein functionally modulates PKC‐mediated phosphorylation and sensitization of the TRPV1 receptor in rat sensory neurons, suggesting the scaffolding protein to be an integral regulator of peripheral inflammatory hyperalgesia.

A non-invasive system for delivering neural growth factors across the blood-brain barrier: A review

Intraventricular administration of nerve growth factor (NGF) in rats has been shown to reduce age-related atrophy of central cholinergic neurons and the accompanying memory impairment, as well as protect these neurons against a variety of perturbations. Since neurotrophins do not pass the blood-brain barrier (BBB) in significant amounts, a non-invasive delivery system for this group of therapeutic molecules needs to be developed. We have utilized a carrier system, consisting of NGF covalently linked to an anti-transferrin receptor antibody (OX-26), to transport biologically active NGF across the BBB. The biological activity of this carrier system was tested using in vitro bioassays and intraocular transplants; we were able to demonstrate that cholinergic markers in both developing and aged intraocular septal grafts were enhanced by intravenous delivery of the OX-26-NGF conjugate. In subsequent experiments, aged (24 months old) Fischer 344 rats received intravenous injections of the OX-26-NGF conjugate for 6 weeks, resulting in a significant improvement in spatial learning in previously impaired rats, but disrupting the learning ability of previously unimpaired rats. Neuroanatomical analyses showed that OX-26-NGF conjugate treatment resulted in a significant increase in cholinergic cell size as well as an upregulation of both low and high affinity NGF receptors in the medial septal region of rats initially impaired in spatial learning. Finally, OX-26-NGF was able to protect striatal cholinergic neurons against excitotoxicity and basal forebrain cholinergic neurons from degeneration associated with chemically-induced loss of target neurons. These results indicate the potential utility of the transferrin receptor antibody delivery system for treatment of neurodegenerative disorders with neurotrophic substances.

Tumor necrosis factor α enhances the sensitivity of rat trigeminal neurons to capsaicin

Tumor necrosis factor alpha (TNFalpha), a pro-inflammatory cytokine, enhances the development of pain and hyperalgesia, although the molecular mechanisms are not well understood. This study evaluated the hypothesis that TNFalpha increases the sensitivity of rat trigeminal neurons to capsaicin via two different mechanisms triggered by either brief or sustained exposure to the cytokine. A brief (5 min) application of TNFalpha significantly sensitized capsaicin-evoked accumulation of intracellular calcium ([Ca2+]i) (226.4+/-37.7 nM vs. 167.5+/-31.3 nM) and increased capsaicin-evoked nocifensive behavior (78.3+/-9.7 vs. 30.9+/-3.6 s) as compared with vehicle pretreatment (P<0.01 for both). Sustained (30 min to 4 h) exposure of cultured neurons to TNFalpha evoked a twofold increase in mRNA transcript (P<0.05) and protein levels (P<0.01) of transient potential receptor vanilloid type 1 (TRPV1). This long-term up-regulation of TRPV1 expression by TNFalpha correlated with enhancement in capsaicin-induced calcitonin gene-related peptide release (P<0.05). Demonstration of colocalization of TNFalpha receptor subtypes I and II with TRPV1 in almost all (>90%) TRPV1 expressing neurons provides evidence consistent with a direct interaction on the same subpopulation of sensory neurons. In summary, our data demonstrate that TNFalpha directly enhances the sensitivity of rat trigeminal neurons to capsaicin via both rapid, non-genomic mechanisms as well as sustained genomic regulation in TRPV1 expression. Thus, increased sensitization and up-regulation of TRPV1 constitutes a potential mechanism by which TNFalpha mediates inflammatory hyperalgesia and pain.