Thesis (M.S.)--West Virginia University, 2009. / Title from document title page. Document formatted into pages; contains vii, 43 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 31-41).

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 6 uppsatser.

Thesis (M.S.)--Miami University, Dept. of Zoology, 2004. / Title from first page of PDF document. Includes bibliographical references (p. 36-47).

Indiana University-Purdue University Indianapolis (IUPUI) / Nerve growth factor (NGF) is a neurotrophin upregulated with injury and inflammation. Peripheral administration of NGF causes hyperalgesia and allodynia in animals. Blocking NGF signaling reverses these effects. At the cellular level, chronic exposure of sensory neurons to NGF enhances expression the neurotransmitter, calcitonin gene-related peptide (CGRP). Acute exposure to NGF increases capsaicin-evoked CGRP release from sensory neurons in culture. Thus, NGF increases peptide release from neurons by: (1) increasing expression of peptides, and/or (2) altering their sensitivity. The increase in peptide outflow by either mechanism could contribute to development of hyperalgesia and allodynia. The signaling cascades mediating the actions of NGF in sensory neurons are unclear. Therefore, experiments were designed to determine which pathways regulate changes in iCGRP content and evoked release from primary sensory neurons in culture. The Ras/MEK/ERK cascade was identified as a possible regulator of iCGRP expression in response to NGF. To test this pathway, it was manipulated in neurons by (1) expression of dominant negative or constitutively active isoforms of Ras, (2) farnesyltransferase inhibition, (3) manipulation of the RasGAP, synGAP, and (4) blocking MEK activity. When the pathway was blocked, the NGF-induced increase in iCGRP expression was attenuated. When the Ras pathway was activated, iCGRP expression increased. These data indicate that Ras, and downstream signaling kinases, MEK and ERK, regulate the NGF-induced increases in CGRP in sensory neurons. To determine which pathway(s) regulate the increase in capsaicin-evoked iCGRP release upon brief exposure to NGF, the Ras/MEK/ERK pathway was manipulated as described above, and pharmacological inhibitors of the PI3 kinase, PLC, and Src kinase pathways were used. There were no differences observed in NGF-sensitization when the Ras and PI3 kinase pathways were inhibited, suggesting these two pathways were not involved. However, when the Src kinase inhibitor PP2 was used, the NGF-induced increase in release was completely blocked. Furthermore, the PKC inhibitor, BIM, also inhibited the sensitization by NGF. This data indicate Src and PKC regulate of sensitivity of sensory neurons in response to brief exposure to NGF. Thus, there is differential regulation of iCGRP content and evoked release from sensory neurons in response to NGF.

The overall objective was to elucidate the mechanism of action of ovulation-inducing factor/nerve growth factor (OIF/NGF) in the reproductive function of spontaneous and induced ovulators, using cow and llama as models. In Study 1, the dynamics of trkA, the high affinity receptor for OIF/NGF, were studied during periovulatory period in cows. Unilateral ovariectomies were performed by colpotomy on Days 2, 4 and 6 of the estrous cycle (Day 0= ovulation), and before and after LH administration. Ovarian samples were processed for immunofluorescent detection of trkA. The intensity and area of immuno-positive staining, and the proportion of immuno-positive cells in both the granulosa and theca layers were higher in dominant than in subordinate follicles (P<0.05). Dominant follicles displayed a different intracellular distribution of trkA from subordinate follicles. The number of positive cells was higher in the developing CL (Day 2 and 4) than in the mature or regressing CL (Day 6, Pre-LH, and Post-LH). In Study 2, the distribution of GnRH neurons in the hypothalamus was examined in female llamas (n = 4). Hypothalamic samples were processed for immunohistochemistry for GnRH. The distribution of GnRH neurons had no evident accumulation in specific hypothalamic nuclei. The majority of GnRH neurons were detected in the anterior and medio-basal hypothalamus (P<0.05). The GnRH neuron fibers were detected primarily in the median eminence and in the medio-basal hypothalamus. In Study 3, the relationship between trkA and GnRH neurons in the llama diencephalon was examined in llama brains (n = 4) obtained in Study 2. Samples were stained using double immunofluorescence. TrkA immuno-reactivity was present in most hypothalamic areas examined; the highest density was found in the diagonal band of Broca and the periventricular nuclei. A low percentage of GnRH cells (1%) showed immuno-reactivity to trkA. Close association between immuno-reactive cells (i.e., GnRH and trkA in the same microscopic field) was detected rarely (3/160 GnRH neurons). We concluded that: 1) the high affinity receptor for OIF/NGF is expressed in greater quantities in dominant than subordinate follicles and in the developing CL; 2) GnRH neurons of llamas are concentrated in the anterior and middle hypothalamus, in close relationship to the third ventricle; and, 3) expression of trkA receptors on GnRH neurons was rare, suggesting that the ovulatory effect of OIF/NGF is not via direct interaction with GnRH neurons.

Creating effective cellular interfaces that can provide specific cellular signals is important for a number of fields ranging from tissue engineering to biosensors. Electroconducting polymers, especially polypyrrole (PPy), have attracted much attention for use in numerous biomedical applications since they provide a potential platform for local delivery of electrical stimuli to target tissues. To effectively modulate cellular functions at neural interfaces, it is essential to incorporate a range of extracellular cues into conducting polymers according to specific applications, such as nerve guidance conduits and implantable neural probes. For nerve regeneration scaffolds, three dimensional forms are desired for control of critical properties, such as porosity, mechanical strength, and topography. However, most researchers have worked on conventional two-dimensional PPy films, which cannot mimic a native three-dimensional architecture. Thus, a portion of my work has focused on introducing three-dimensional nanofibrous features into PPy. I have investigated various coating conditions to obtain uniform and conductive nanofibers. Effectiveness of electrical stimulation through the conducting nanofibers was confirmed by in vitro PC12 cell culture. The effects of different conducting nanofiber topographies (random and aligned) on cell adhesion and neurite outgrowth were examined in conjunction with electrical stimulation. The benefits of immobilized-NGF could be combined with electrical stimuli, which could be an ideal platform for neural tissue engineering scaffolds. Thus, I have modified conducting polymers to display neurotrophic activity. Nerve growth factor (NGF) was chemically immobilized on two dimensional and three dimensional PPy substrates. Specific chemical conjugation was achieved and characterized using diverse techniques. Immobilized NGF was as effective as exogenous NGF in medium in inducing neurite development and extension. NGF immobilized on functionalized PPy substrates was stable in a physiological solution and under electrical stimulation, indicating effective prolonged activity. I also investigated another important application of conducting polymer-based materials for neural interfacing - passivating electrodes with a biocompatible polysaccharide, hyaluronic acid (HA). I synthesized electrically polymerizable HA by chemically conjugating amine-functionalized pyrrole derivatives with HA. This coating was stable under physiological conditions for three months and resistant to enzymatic degradation. In vitro studies have shown the minimal adhesion and migration of astrocytes on the HA-coated electrodes. Implantation of HA-coated commercial probes into rat cortices for three weeks revealed attenuated reactive astrocyte responses from the coated wires, and the importance of glial interaction with non-conducting sites was demonstrated. / text

Lai, Ching Janice. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 148-168). / Abstracts in English and Chinese. / Abstract --- p.i / Abstract in Chinese --- p.iii / Acknowledgements --- p.v / Table of Content --- p.vi / List of Tables --- p.x / List of Figures --- p.xi / Abbreviations --- p.xii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- General Introduction --- p.1 / Chapter 1.2 --- Nervous System and the Blood-Brain-Barrier --- p.3 / Chapter 1.3 --- Glial cells --- p.3 / Chapter 1.4 --- Studying Astrocyte Responses As a New Direction in Neuroscience --- p.4 / Chapter 1.5 --- The Roles of Astrocyte in the CNS --- p.5 / Chapter 1.5.1 --- Energy-Dependent Communication Between Neurons and Astrocytes --- p.7 / Chapter 1.5.2 --- Strategies for Metabolic Exchange Between Astrocytes and Neurons --- p.8 / Chapter 1.5.2.1 --- Provision of Energy Metabolites to Neurons by Astrocytes --- p.9 / Chapter 1.5.2.2 --- Glucose Transporters in the CNS --- p.10 / Chapter 1.5.2.3 --- The Lactate Shuttle Hypothesis --- p.12 / Chapter 1.5.2.4 --- The Regulation of Glucose Uptake at the Blood-Brain-Barrier (BBB) by the Activity of Neurons --- p.14 / Chapter 1.5.3 --- Alternation of Energy Metabolism in Neuropathy --- p.15 / Chapter 1.5.3.1 --- Ketone Body Shuttle Hypothesis --- p.15 / Chapter 1.5.3.2 --- The Utilization of Free Fatty Acids by the Brain --- p.17 / Chapter 1.5.4 --- The Provision of Neurotrophic Factors to Neurons by Astrocytes --- p.17 / Chapter 1.5.4.1 --- Neurotrophins --- p.18 / Chapter 1.5.4.1.1 --- Relationship Between Neurotrophins and Glucose --- p.20 / Chapter 1.5.4.2 --- S100B --- p.21 / Chapter 1.5.5 --- Astrocytic Cholesterol in Astrocytes as a Neurotrophic Factor --- p.22 / Chapter 1.6 --- Neuroprotective Effect of Glucose vi - --- p.23 / Chapter 1.7 --- Diseases Associated with Decreased Glucose Transport at the BBB --- p.24 / Chapter 1.7.1 --- Glucose Transporter Type 1 Deficiency Syndrome (GlutlDS) --- p.24 / Chapter 1.7.2 --- Hypoglycemia with Insulin Therapy for Diabetes Patients --- p.24 / Chapter 1.8 --- Aims and Hypothesis of Study --- p.26 / Chapter Chapter 2. --- 2 Materials and Methods --- p.27 / Chapter 2.1 --- Materials --- p.27 / Chapter 2.1.1 --- Cell Culture --- p.27 / Chapter 2.1.1.1 --- Cells --- p.27 / Chapter 2.1.1.1.1 --- C6 cells --- p.27 / Chapter 2.1.1.1.2 --- Primary Astrocytes --- p.27 / Chapter 2.1.1.2 --- Cell Culture Reagent --- p.27 / Chapter 2.1.2 --- Study of Growth Properties --- p.31 / Chapter 2.1.2.1 --- Equipment for Growth Curve Construction --- p.31 / Chapter 2.1.2.2 --- Reagents for Flow Cytometry --- p.32 / Chapter 2.1.2.3 --- Reagents for 3H-thymidine Incorporation Assay --- p.32 / Chapter 2.1.3 --- Study of Neurotrophic Properties --- p.33 / Chapter 2.1.3.1 --- Determination of Neurotrophic Factor Productions --- p.33 / Chapter 2.1.3.1.1 --- Reagents and Buffers for Northern Blot Analysis --- p.33 / Chapter 2.1.3.2 --- Reagents and Buffers for Western Blot Analysis --- p.43 / Chapter 2.1.3.2.1 --- Protein Assay --- p.43 / Chapter 2.1.3.2.2 --- Reagents for SDS Polyacrylamide Electrophoresis of Proteins --- p.44 / Chapter 2.1.3.2.3 --- Reagents for the Transfer of Protein to Membrane and Signal Detection --- p.47 / Chapter 2.1.4 --- Study of Lipid in Glial cells --- p.50 / Chapter 2.1.4.1 --- Determination of Genes Expression in Lipid Metabolism --- p.50 / Chapter 2.1.4.2 --- Reagents for Determination of Cholesterol and Fatty Acid Levels by Gas Chromatography --- p.50 / Chapter 2.2 --- Methods --- p.54 / Chapter 2.2.1 --- Cell culture --- p.54 / Chapter 2.2.1.1 --- Maintenance of C6 cells --- p.54 / Chapter 2.2.1.2 --- Primary Culture of Rat Astrocytes --- p.54 / Chapter 2.2.2 --- Study of Growth Properties of Glial Cells vii - --- p.56 / Chapter 2.2.2.1 --- Construction of cell growth curve --- p.56 / Chapter 2.2.2.2 --- Flow Cytometric Analysis of Cell Cycle Profile --- p.56 / Chapter 2.2.2.3 --- Measurement of DNA Synthesis --- p.57 / Chapter 2.2.3 --- Study of Neurotrophic Properties --- p.58 / Chapter 2.2.3.1 --- Determination of Neurotrophic Facotor Production --- p.58 / Chapter 2.2.3.1.1 --- Northern Blot Analysis --- p.58 / Chapter 2.2.3.1.2 --- Western Blot Analysis --- p.67 / Chapter 2.2.3.2 --- Determination of Gene Expression in Lipid Metabolism --- p.72 / Chapter 2.2.3.2.1 --- Northern Blot Analysis --- p.72 / Chapter 2.2.3.2.2 --- RT-PCR --- p.72 / Chapter 2.2.3.3 --- Study of Lipid Profiles in Glial Cells --- p.73 / Chapter 2.2.3.3.1 --- Sample preparation --- p.73 / Chapter 2.2.3.3.2 --- Total Cholesterol Determination --- p.73 / Chapter 2.2.3.3.3 --- Total Fatty Acid Determination --- p.75 / Chapter 2.2.3.3.4 --- Quantification of Proteins --- p.76 / Chapter 2.2.4 --- Statistical Analysis --- p.77 / Chapter Chapter 3 --- Results --- p.78 / Chapter 3.1 --- The effects of glucose deficiency on cell proliferation --- p.78 / Chapter 3.1.1 --- Direct Cell Count Assay --- p.78 / Chapter 3.1.2 --- Flow Cytometry Assay --- p.83 / Chapter 3.1.3 --- 3H-Thymidine Uptake Assay --- p.85 / Chapter 3.2 --- The Effects of Glucose Deficiency on Neurotrophic Properties of Glial Cells --- p.87 / Chapter 3.2.1 --- The Effects of Glucose Deficiency on mRNA and Protein Expressions of Neurotrophins --- p.88 / Chapter 3.2.1.1 --- Northern Blot Assays --- p.88 / Chapter 3.2.1.2 --- Western Blot Assays --- p.93 / Chapter 3.2.2 --- The Effects of Glucose Deficiency on Lipid Homeostasis --- p.96 / Chapter 3.2.2.1 --- Northern Blot Assays --- p.96 / Chapter 3.2.2.2 --- Gas Chromatography Assays --- p.101 / Chapter 3.2.2.2.1 --- Cholesterol Analyses --- p.102 / Chapter 3.2.2.2.2 --- Fatty Acid Analyses --- p.105 / Chapter Chapter 4 --- Discussion --- p.115 / Chapter 4.1 --- The in vitro Model of Hypoglycorrhachia --- p.115 / Chapter 4.2 --- Decreased Glucose Level Triggers Changes of Gial Cells Proliferation --- p.117 / Chapter 4.3 --- Expression of Neurotrophic Factor under Glucose Deficiency viii - --- p.120 / Chapter 4.3.1 --- Alteration of the Expression of Neurotrophins --- p.120 / Chapter 4.3.1.1 --- NGF --- p.122 / Chapter 4.3.1.2 --- BDNF --- p.123 / Chapter 4.3.1.3 --- NT-3 --- p.126 / Chapter 4.3.1.4 --- NT-4/5 --- p.128 / Chapter 4.3.2 --- Alteration of the mRNA Expression of Calcium Binding ProteinS100B --- p.128 / Chapter 4.4 --- Alteration of Lipid Metabolism in Decreased Glucose Supply --- p.130 / Chapter 4.4.1 --- Up-regulation of ApoE mRNA Expression in Glucose Deficiency --- p.133 / Chapter 4.4.2 --- Cholesterol Homeostasis in Glial Cells --- p.133 / Chapter 4.4.3 --- Fatty Acids Homeostasis in Glial Cells --- p.135 / Chapter 4.4.4 --- Decreased Ketone Bodies synthesis in Glucose Deficiency --- p.143 / Chapter 4.5 --- Limitations of the Current Study --- p.144 / Chapter 4.6 --- Future Directions --- p.145 / Chapter Chapter 5 --- Conclusion --- p.147 / References --- p.148 / Appendix --- p.169

Peripheral nerve damage affects hundreds of thousands of people every year. This study tested the effectiveness of using lysophosphatidylcholine (LPC) in combination with nerve growth factor (NGF) to increase the healing rate of damaged left sciatic nerves in female rats. The rats were randomly divided into eight groups: Sham, Right Sciatic, Crush, LPC, LPC-NGF, Crush- LPC, Crush-NGF, and Crush-LPC-NGF. The healing of the nerves was measured by monitoring gait, electrophysiological parameters (compound muscle action potential amplitudes and nerve conductance velocities) and morphological parameters (total fascicular area, total myelinated fiber counts, fiber densities, fiber diameters, and g-ratio). Gait and electrophysiological parameters were measured three times a week. Morphological parameters were measured at three weeks and at six weeks. The LPC and LPC-NGF groups were not statistically different from the controls (Sham and Right Sciatic) at either of the morphological time points but were statistically different from the controls for the first three weeks for the electrophysiological parameters and gait. The LPC-NGF group did not differ from the LPC group at any time point for any of the parameters. Crush, Crush-LPC, Crush-NGF, and Crush-LPC-NGF groups statistically differed from the controls at week 3 for all parameters and only in the electrophysiological parameters at week 6. Crush-LPC, Crush-NGF, and Crush-LPC-NGF did not differ from each other or from the Crush group. The combination of LPC and NGF did not prove to be an effective treatment for peripheral nerve damage. Future work is recommended to test multiple injections of LPC and NGF.

Biological responses to orthodontic forces involve various cell types, these include fibroblasts, endothelial cells, blood vessels and sensory nerves in the periodontal ligament as well as osteoblasts, osteoclasts and cementoblasts in roots and bone surfaces. Neurotrophins are believed to interact with these cells to initiate the process of bone resorption particularly during orthodontic tooth movement. Neuropeptides released from sensory neurons have been shown to modulate the tissue inflammatory responses. In addition, neurotrophins, including nerve growth factor (NGF), play an important role in neural cell differentiation and survival. The exact localization and function of neurotrophins and neurotrophic receptors in the dento-alveolar complex remains unclear. Moreover, the identity and distribution of structures expressing neurotrophins and neurotrophic receptors has yet to be fully determined. It is reasonable to propose that periodontal ligament and alveolar bone remodelling may be influenced by NGF. In addition, anti-NGF may block neurochemical changes and, hence, inhibit orthodontic tooth movement. The aims of this research were to investigate the cells responsible for NGF secretion within the periodontal ligament (PDL), pulp and bone, and the effect that anti-NGF might have on orthodontic tooth movement. 28, 8 week-old, male Sprague-Dawley rats were randomly divided into control and experimental groups. Fourteen experimental animals had anti-NGF injected paradentally. Animals were sacrificed at 7 and 14 days. Sections from an earlier study were examined and stained using TRAP for osteoclast identification and analysed histomorphometrically to enable comparisons between control and experimental groups. The findings of this investigation indicated that injections of anti-NGF did not significantly affect the rate of tooth movement with the use of different tooth movement measurement methods. TRAP staining proved to be a useful and reliable marker of osteoclasts. TRAP-positive osteoclastic cells were detected in both anti-NGF and control groups. However, the TRAP-positive cells were not stained intensely with NGF immunolabelling. On the other hand, cells that were stained intensely with NGF, were TRAP-negative. The results suggested that both sympathetic and nociceptive nerves might function in counter balance to modulate bone resorption, and osteoclasts might not be directly responsible for NGF secretion within the PDL and bone. Further studies to determine the effect of NGF on tooth movement are warranted to more clearly identify the NGF expressing cells within the rat dento-alveolar complex and possible role played by NGF in orthodontic tooth movement. / http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1297498 / Thesis (D.Clin.Dent.)-- School of Dentistry, 2007

Herpes simplex viruses (HSV) establish latent infections in sensory neurons of their host and are maintained in this state by little understood mechanisms that, at least in part, are regulated by signalling through nerve growth factor (NGF) and its receptor tropomyosin related kinase, trkA. Previous studies have demonstrated that Zhangfei is a transcriptional factor that is expressed in differentiated neurons and is thought to influence HSV replication and latency. Zhangfei, like the HSV trans-activator VP16 and Luman, binds the ubiquitous nuclear protein host cell factor (HCF) inhibiting the ability of VP16 and Luman to initiate HSV replication. <p>Recently, Brn3a, another neuronal factor thought to influence HSV latency and reactivation was found to possess an HCF-binding domain and could potentially require HCF for activity. The neuronal POU IV domain protein, Brn3a, among its many regulatory functions has been described as an enhancer of the NGF receptor trkA, during development in mouse. I therefore investigated the possible link between Brn3a, TrkA, NGF signaling, HCF, Zhangfei and HSV-1 latency and reactivation. I hypothesized that Zhangfei would also suppress the ability of Brn3a to activate the expression of TrkA and that this would have an impact on NGF-TrkA signaling and, consequently on HSV-1 reactivation from latency.<p>My first study determined which Brn3a/trkA promoter interactions were important for trkA transcription. I constructed a plasmid that contains 1043 base pairs of genomic sequences that extend from 30 nucleotides upstream of trkA coding region. In contrast to previous data, a short 190 bp region that lies proximal to the trkA initiation codon was sufficient for Brn3a trans-activation in NGF-differentiated PC12, Vero and human medulloblastoma cells. At least two portions of the 190 bp fragment bind to Brn3a. In addition, Brn3a increased endogenous levels of trkA transcripts in PC12 cells and initiated trkA expression in medulloblastoma cells, which normally do not express trkA. <p>The second step was to determine the effects of HCF and Zhangfei association with Brn3a on trkA trans-activation. I found that Brn3a required HCF for activating the trkA promoter and that Zhangfei has a suppressive effect over Brn3a-trkA activation in non-neuronal cells. In sympathetic neuron-like NGF-treated PC12 cells, Zhangfei did not suppress the ability of Brn3a to activate the TrkA promoter, however, Zhangfei was able capable of inducing the expression of TrkA in the absence of Brn3a. Both Brn3a and Zhangfei induced the expression of endogenous trkA in PC12 cells.<p>Since Vero and PC12 cells are not from human origin I wanted to examine the ability of Zhangfei to induce trkA transcription in medulloblastoma cells, that because of its tumor nature do not express trkA. TrkA transfections in these cells have shown to drive them to cell arrest or apoptosis. Since Zhangfei is not express in medulloblastoma tumors I then used ONS-76 medulloblastoma cells as a model to determine Zhangfeis envolvement in the NGF-trkA signaling pathway.<p> I show herein that in ONS-76 medulloblastoma cells resveratrol, an inducer of apoptosis and differentiation, increased the expression of Zhangfei and trkA as well as Early Growth Response Gene 1 (Egr1), a gene normally activated by NGF-trkA signalling. ONS-76 cells stop growing soon after treatment with resveratrol and a portion of the cell undergo apoptosis. While the induction of Zhangfei in resveratrol-treated cells was modest albeit consistent, the infection of actively growing medulloblastoma cells with an adenovirus vector expressing Zhangfei mimicked the effects of resveratrol. Zhangfei activated the expression of trkA and Egr1 and caused these cells to display markers of apoptosis. The phosphorylation of Erk1, an intermediate kinase in the NGF-trkA signaling critical for differentiation, was observed in Zhangfei infected cells, supporting the hypothesis that Zhangfei is a mediator of trkA-NGF signaling in theses cells leading either to differentiation or apoptosis. Binding of HCF by Zhangfei did not appear to be required for this effect as a mutant of Zhangfei incapable of binding HCF was also able to induce the expression of trkA and Egr1. <p>In in vivo and in vitro models of HSV-1 latency, the virus reactivates when NGF supply to the neuron is interrupted. Based on the above evidence Zhangfei, in HSV-1 latently infected neurons, would have the ability to prolong a state of latency by inducing trkA expression allowing the activation of NGF-trkA signaling pathway. Since NGF is produced by many cell types it is possible that reactivation is triggered not by a decrease in NGF but by a down-regulation of TrkA expression.Therefore, if Zhangfei expression is suppress the trkA signaling could be interrupted or shifted towards apoptosis signaling, this would allow neuronal HCF-binding proteins like Luman, which can activate HSV IE expression, to initiate HSV IE expression and subsequently viral replication.