Um bom modelo experimental deve contar com métodos de avaliação eficazes de seus parâmetros. Esta é uma observação importante quando se faz necessária a avaliação da nocicepção e da incapacitação articular em animais experimentais. O estabelecimento de novos critérios aos testes animais é fundamental para que processos inflamatórios articulares possam continuar sendo estudados, entendidos e resolvidos. Buscando contribuir neste sentido, este trabalho realizou a padronização do teste de incapacitação dinâmico (TID) para avaliação da incapacitação articular em modelos experimentais de artrite. Os resultados obtidos mostram que o TID é sensível na avaliação da incapacitação articular em modelos de artrite induzida por antígeno (AIA) ou por zimosana. Além disso é preditivo para o estudo do efeito farmacológico de drogas que interfiram na incapacitação articular como anti-inflamatórias ou analgésicas. Desde o início da década de 90, quando participação das células da glia na dor foi descrita, diversos trabalhos surgiram mostrando seu papel em diferentes modelos animais. A participação das células da glia espinais na dor e incapacitação em modelos experimentais de artrite e artrite reumatoide têm sido relatada, mas não há descrição desta participação em função do tempo de indução do processo inflamatório articular. Por meio de ferramentas farmacológicas e moleculares, este trabalho mostra que as células da glia, tanto espinais como do gânglio da raiz dorsal estão participando na gênese e manutenção da incapacitação inflamatória articular em modelo de AIA. A participação destas células ocorre por meio da liberação de IL-1? e TNF? em nível medular e pela primeira vez é mostrado que a ativação astrocítica parece preceder a ativação microglial neste modelo. / A good experimental model must rely on effective methods of evaluation of its parameters. This is an important observation when it is necessary to evaluate the articular nociception and disability in experimental animals. Establishing new criteria to test animals is essential for inflammatory joint can continue being studied, understood and resolved. Seeking help in this sense, this work constitutes a test dynamic weight bearing (DWB) standardization for assessment of articular incapacitation in experimental models of arthritis. The results show that the DWB is sensitive in assessing the impairment models articular antigen-induced arthritis (AIA) or zimosana. Furthermore is predictive for studying the pharmacological effects of drugs that interfere with articular incapacitation as antiinflammatory or analgesic. Since the early 90s, when participation of glial cells in pain was described, several studies have emerged showing its role in different animal models. The involvement of glial cells in the spinal pain and disability in experimental models of arthritis and rheumatoid arthritis have been reported, but no description of this contribution versus time of induction of joint inflammation. Through molecular and pharmacological tools, this work shows that the glial cells, both as the spinal dorsal root ganglio are participating in the genesis and maintenance of inflammatory joint incapacitation in AIA model. The participation of these cells occurs through the release of IL-1? and TNF? in the spinal cord and the first time it is shown that astrocytic activation appears to precede the microglial activation in this model.

Fetal alcohol spectrum disorders (FASD) are caused by alcohol exposure during pregnancy and is the leading cause of mental retardation. Alcohol exposure during development results in the loss of neurons in the developing brain. The underlying molecular mechanisms are unclear and there currently is no cure for FASD. Ethanol-induced neuronal death is accompanied by neuroinflammation. Chemokine monocyte chemoattractant protein 1 (MCP-1) and its receptor C-C chemokine receptor type 2 (CCR2) are critical mediators of neuroinflammation and microglial activation. Using a third trimester equivalent mouse model of ethanol exposure, we found that treatment of Bindarit (MCP-1 synthesis inhibitor) and RS504393 (CCR2 antagonist) significantly reduced ethanol-induced microglia activation/neuroinflammation, and neuroapoptosis in the developing brain. Moreover, ethanol plus MCP-1 caused more neuronal death in a neuron/microglia co-culture system than neuronal culture alone, and Bindarit and RS504393 attenuated ethanol-induced neuronal death in the co-culture system. Ethanol activated TLR4 and GSK3β, two key mediators of microglial activation in the brain and cultured microglial cells (SIM-A9). Blocking MCP-1/CCR2 signaling attenuated ethanol-induced activation of TLR4 and GSK3β. Further, we determined whether knocking out of MCP-1/CCR2 ameliorates neonatal alcohol exposure-induced long-lasting behavioral deficits in adolescent and adult mice. C57BL/6 and MCP-1-/-/CCR2-/- mice were exposed to alcohol (5 g/kg) by subcutaneously injection on PD4. A series of behavioral tests including Open Field (PD 35-36 and PD 70-71), Rotor-Rod (PD 38 and PD 73), Balance Beam (PD 40 and PD75) and Morris Water Maze (PD 42 and PD77) were performed in the adolescence and adulthood. We found that MCP-1-/-/CCR2-/- mice were resistant to neonatal alcohol exposure-induced deficits in motor function in the Rotor-Rod and Balance Beam tests; MCP-1 and CCR2 deficiency also protected mice against neonatal ethanol exposure induced long lasting deficits in learning and memory in the Morris Water Maze testing. Collectively, these results suggest that MCP-1/CCR2 signaling plays an important role in ethanol-induced microglial activation/neuroinflammation and neurodegeneration in the developing brain and also plays an important role in developmental alcohol exposure induced long-lasting behavioral deficits in adolescence and adulthood.

Parkinson’s disease is a progressive neurodegenerative disorder, characterized by a depletion of the dopaminergic neurons in the substantia nigra. The cause of the disease is yet unknown but age, oxidative stress, and neuroinflammation are some of the features involved in the degeneration. In addition, substantial cell death of noradrenergic neurons occurs in the locus coeruleus (LC). Noradrenaline has been suggested to protect the dopamine neurons from oxidative stress and neuroinflammation. The main treatment of Parkinson’s disease is Levo-dopa, although severe side effects arise from this therapy. Hence, grafting fetal ventral mesencephalic (VM) tissue into the adult striatum has been evaluated as an alternative treatment for Parkinsons’s disease. However, the survival of the grafted neurons is limited, and the dopamine-denervated striatum does not become fully reinnervated. Therefore, elucidating factors that enhance dopamine nerve fiber formation and/or survival of the grafted neurons is of utmost importance. To investigate dopamine nerve fiber formation and the interactions with glial cells, organotypic VM tissue cultures were utilized. Two morphologically different nerve fiber outgrowths from the tissue slice were observed. Nerve fibers were initially formed in the absence of migrating astrocytes, although thin vimentin-positive astrocytic processes were detected within the same area. A second, persistent nerve fiber outgrowth was observed associated with migrating astrocytes. Hence, both of these nerve fiber outgrowths were to some extent dependent on astrocytes, and appeared as a general feature since this phenomenon was demonstrated in β-tubulin, tyrosine hydroxylase (TH), and aldehyde dehydrogenase A1 (ALDH1)-positive nerve fibers. Neither oligodendrocytes (NG2-positive cells), nor microglia (Iba-1-positive cells) exerted any effect on these two neuronal growths. Since astrocytes appeared to influence the nerve fiber formation, the role of proteoglycans, i.e. extracellular matrix molecules produced by astrocytes, was investigated. β-xyloside was added to the cultures to inhibit proteoglycan synthesis. The results revealed a hampered astrocytic migration and proliferation, as well as a reduction of the glia-associated TH-positive nerve fiber outgrowth. Interestingly, the number of cultures displaying the non-glia-mediated TH-positive nerve fibers increased after β-xyloside treatment, although the amount of TH-protein was not altered. Thus, proteoglycans produced by astrocytes appeared to be important in affecting the dopamine nerve fiber formation. The noradrenaline neurons in LC have been suggested to protect dopamine neurons from damage. Therefore, the interaction between VM and LC was evaluated. Using the intraocular grafting method, fetal VM and LC were grafted either as single grafts or as VM+LC co-grafts. Additionally, the recipient animals received 2% blueberry-enriched diet. The direct contact of LC promoted graft volume and survival of TH-positive neurons in the VM grafts. The number of dopamine neurons, derived preferably from the A9 (ALDH1/TH-positive) was increased, whereas the dopamine neurons from the A10 (calbindin/TH-positive) were not affected. A dense dopamine-β-hydroxylase (DBH)-positive innervation was correlated to the improved survival. Blueberry-enriched diet enhanced the number of TH-positive neurons in VM, although the graft size was not altered. The combination of blueberries and the presence of LC did not yield additive effects on the survival of VM grafts. The attachment of VM or the addition of blueberries did not affect the survival of TH-positive neurons in LC grafts. The number of Iba-1-positive microglia was decreased in co-grafted VM compared to single VM transplants. The addition of blueberries reduced the number of Iba-1-positive microglia in single VM transplants. Hence, the direct contact of LC or the addition of blueberries enhanced the survival of VM grafts. Taken together, these data demonstrate novel findings regarding the importance of astrocytes for the nerve fiber formation of dopamine neurons. Further, both the direct attachment of LC or antioxidant-enriched diet promote the survival of fetal VM grafts, while LC is not affected.

Reproductive function is essential to the survival of all species. In mammals and other vertebrates, the control of reproduction relies on the hypothalamic-pituitary-gonadal axis, with the primary driving force provided by hypothalamic GnRH neurons. In the median eminence, the decapeptide GnRH are released in a unique pattern from GnRH neuroterminals into the portal capillary system as part of reproductive cycle. During aging, the biological rhythms of GnRH release are altered in a species-specific manner, with a reduction of GnRH pulsatility and surge in aging female rats resulting in reproductive senescence, which happens much earlier than gonadal failure in rats. Relatively few studies have focused on regulation of GnRH release at the neuroterminal level in the median eminence during reproductive aging. Therefore, the aims of this dissertation are to 1) Study the regulation of GnRH secretion at the neuroterminal level, focusing on glutamate transmission; 2) Ascertain the interaction between GnRH neuroterminals and their surrounding microenvironment focused on glial cells and the portal capillary system in the median eminence; and 3) Analyze age and hormone effects on GnRH neuroterminals and their microenvironment. An aging ovariectomized female rat model was used to study the effects of age and hormones on GnRH neuroterminal system. Fluorescence microscopy, confocal microscopy and transmission electron microscopy were used in conjunction with several imaging analysis tools. I mastered the use of cryo-embedding multi-probe immunogold labeling electron microscopy, which was essential to visualize and quantify the ultrastructral changes in GnRH neuroterminals. I combined the serial electron microscopy with cryo-embedding immunogold electron microscopy preparation and developed a new technique to examine biological markers with a three-dimensional perspective at the cellular level. Results from a series of four research projects showed: 1) There is a novel glutermatergic pathway in GnRH neuroterminals, which may regulate GnRH secretion; 2) There are dramatic age related morphological changes in the GnRH neuroterminal /glia/ portal capillary system of the median eminence that may be involved in reproductive senescence and other neuroendocrine system impairments with age; 3) Serial electron microscopy combined with immunogold labeling technique is a useful method to study the regulation of neuronal signaling pathway. Although my studies were performed on a rat model, it seems reasonable to predict that some of these changes in the median eminence with age may apply to other species, including humans, relevant to some of the menopausal symptoms in women. / text

Neurogenesis occurs in exclusive regions in the adult nervous system, the subventricular zone and dentate gyrus in the brain, and olfactory epithelium (OE) in the periphery. Cell replacement after death or injury, occurs to varying degrees in neural tissue, and is thought to be dependent upon the biological responses of stem and/or progenitor cells. Despite the progress made to identify adult OE and central nervous system (CNS) progenitors and lineage trace their progeny, our spatial and temporal understanding of embryonic OE neuroglial progenitors has been stalled by the paucity of identifiable genes able to distinguish individual candidate progenitors. In the developing CNS, radial glia serve as both neural progenitors and scaffolding for migrating neuroblasts and are identified by the expression of a select group of antigens, including nestin. Here, I show that the embryonic OE contains a novel radial glial-like progenitor (RGLP) that is not detected in adult OE. RGLPs express the radial glial antigens nestin, GLAST and RC2, but not brain lipid binding protein (BLBP), which, distinct from CNS radial glia, is instead found in olfactory ensheathing cells, a result confirmed using lineage tracing with BLBP-cre mice. Nestin-cre-mediated lineage tracing with three different reporters reveals that only a subpopulation of nestin-expressing RGLPs activate the “CNS-specific” nestin regulatory elements, and produce spatially restricted neurons in the OE and vomeronasal organ. The dorsal-medial restriction of transgene-activating cells is also seen in the embryonic OE of Nestin-GFP transgenic mice, where GFP is found in a subpopulation of GFP+ Mash1+ neuronal progenitors, despite the fact that endogenous nestin expression is found in RGLPs throughout the OE. In vitro, embryonic OE progenitors produce three biologically distinct colony subtypes, that when generated from Nestin-cre/ZEG mice, produce GFP+ neurons, recapitulating their in vivo phenotype, and are enriched for the most neurogenic colony subtype. Neurogenesis in vitro is driven by the proliferation of nestin+ progenitors in response to FGF2. I thus provide evidence for a novel neurogenic precursor, the RGLP of the OE, that can be regulated by FGF2, and provide the first evidence for intrinsic differences in the origin and spatiotemporal potential of distinct progenitors during OE development.

Neuronal-glial interactions play an important role in the development of neuropathic (NP) pain states. Earlier studies in our laboratory suggest a role for activated glia in morphine-induced delta opioid receptor (DOR) trafficking by altering DOR functional competence. Thus, chronic treatment with the glial inhibitor, propentofylline (PF) blocks the anti-allodynic and anti-hyperalgesic effects of the DOR agonist deltorphin II. The present study aimed to determine whether NP pain-induced changes in DOR function and trafficking are dependent on glial activation. The first global aim of this study was to determine the molecular and behavioural effects of glial activation by two glial inhibitors, PF and PJ34 in a model of neuropathic pain. Glial activation was assessed via changes in specific proteins using fluorescent immunohistochemistry (IHC). Neuropathy-induced c-Fos activation was assessed by IHC and pain hypersensitivity was assessed, including mechanical allodynia and spontaneous pain. The second global aim determined the role of activated glia in changes in neuropathy-induced increases in DOR function and DOR subcellular localization using immunogold IHC and transmission electron microscopy (EM). Chronic PJ34 attenuated chronic constriction injury (CCI)-induced mircoglial, but not astrocyte activation. Chronic administration of either PF or PJ34 attenuated the CCI-induced increase in c-Fos immunoreactive expression. However, neither drug attenuated CCI-induced mechanical allodynia or spontaneous pain. Both chronic PF and PJ34 administration in NP animals attenuated the anti-allodynic effects of the DOR-selective agonist deltorphin II, suggesting glial inhibition blocks DOR function. However, chronic PF, but not PJ34, blocked the anti-allodynic effects of another DOR agonist, SNC80. These data suggest that SNC80 might be targeting a different DOR molecular species that is not affected by factors released from microglia. Finally, EM experiments revealed that chronic PF treatment prevented the CCI-induced increase in DOR trafficking providing a positive correlation between behaviour and receptor localization. This study suggests that activated glia contribute to changes in DOR function and trafficking in NP pain states. It also suggests that there is a dissociation between glial inhibition and pain hypersensitivity. / Thesis (Master, Pharmacology & Toxicology) -- Queen's University, 2010-08-31 14:45:47.888

Neurogenesis occurs in exclusive regions in the adult nervous system, the subventricular zone and dentate gyrus in the brain, and olfactory epithelium (OE) in the periphery. Cell replacement after death or injury, occurs to varying degrees in neural tissue, and is thought to be dependent upon the biological responses of stem and/or progenitor cells. Despite the progress made to identify adult OE and central nervous system (CNS) progenitors and lineage trace their progeny, our spatial and temporal understanding of embryonic OE neuroglial progenitors has been stalled by the paucity of identifiable genes able to distinguish individual candidate progenitors. In the developing CNS, radial glia serve as both neural progenitors and scaffolding for migrating neuroblasts and are identified by the expression of a select group of antigens, including nestin. Here, I show that the embryonic OE contains a novel radial glial-like progenitor (RGLP) that is not detected in adult OE. RGLPs express the radial glial antigens nestin, GLAST and RC2, but not brain lipid binding protein (BLBP), which, distinct from CNS radial glia, is instead found in olfactory ensheathing cells, a result confirmed using lineage tracing with BLBP-cre mice. Nestin-cre-mediated lineage tracing with three different reporters reveals that only a subpopulation of nestin-expressing RGLPs activate the “CNS-specific” nestin regulatory elements, and produce spatially restricted neurons in the OE and vomeronasal organ. The dorsal-medial restriction of transgene-activating cells is also seen in the embryonic OE of Nestin-GFP transgenic mice, where GFP is found in a subpopulation of GFP+ Mash1+ neuronal progenitors, despite the fact that endogenous nestin expression is found in RGLPs throughout the OE. In vitro, embryonic OE progenitors produce three biologically distinct colony subtypes, that when generated from Nestin-cre/ZEG mice, produce GFP+ neurons, recapitulating their in vivo phenotype, and are enriched for the most neurogenic colony subtype. Neurogenesis in vitro is driven by the proliferation of nestin+ progenitors in response to FGF2. I thus provide evidence for a novel neurogenic precursor, the RGLP of the OE, that can be regulated by FGF2, and provide the first evidence for intrinsic differences in the origin and spatiotemporal potential of distinct progenitors during OE development.

[Truncated abstract] Olfactory ensheathing glia (OEG) have been demonstrated to improve functional and anatomical outcomes after injury to the nervous system and are currently being trialled clinically. This thesis presents the investigation of two important issues in OEG biology. The first study (Chapter 2) investigates effects of different members of the neuregulin (NRG) family of molecules on the proliferation of OEG, as a means of quickly obtaining large numbers of cells for clinical or experimental use. We report that NRG-1β, but not NRG- 2α or NRG-3, has a significant proliferative effect. Furthermore, we report for the first time that use of different mitogens (forskolin and pituitary extract) commonly used to expand these cells in vitro, can have a significant effect on the responsiveness of OEG to added NRG in subsequent mitogenic assays. OEG grown initially with forskolin and pituitary extract exhibited increased basal proliferation rates in comparison to OEG originally expanded without these factors, and this increased rate of proliferation was sustained for at least 6 days following their withdrawal from the culture medium. We also report for the first time the expression pattern of ErbB2, ErbB3 and ErbB4 receptors on p75-selected OEG, and investigate their contribution to the NRG mitogenic effect by the use of inhibitory ErbB antibodies. Our second study (Chapter 3) seeks to clarify the role of OEG in promoting myelination of central nervous system neurons. In this study we have investigated the myelinating ability of OEG derived from embryonic (EEG), postnatal (PEG) and adult tissue (AEG) both in vitro and in vivo. OEG selected by p75-immunopanning were co-cultured with dissociated cultures of TrkA-dependant embryonic dorsal root ganglion (DRG) neurons. EEG, but not AEG or PEG, successfully myelinated DRG neurons in the presence of serum and/or ascorbate. AEG also failed to myelinate GDNF-dependant embryonic DRG cultures, and growth factor-independent adult DRG cultures. Transplantation of OEG into lysolecithin demyelinated spinal cord demonstrated distinct ultrastructural differences between transplants of OEG derived from animals of different ages. Furthermore, we demonstrate that clearance of degraded myelin from the lesion site appears to be more effective when animals are transplanted with EEG rather than AEG or Schwann cell preparations. These results suggest that myelinating potential of OEG in vitro and behaviour of these cells following transplantation in vivo are developmentally regulated.

Thesis (M.S.)--Uniformed Services University of the Health Sciences, 2006 / Typescript (photocopy)

The topic of the thesis is the mechanisms of cellular volume regulation in the rat retina. Müller cells as main macroglial cells of the retina are supposed to play important roles in the regulation of the retinal ion- and osmohomeostasis and, thus, in the regulation of the extracellular space volume. In the first part of the thesis, signaling pathways were determined which are involved in the regulation of the volume of Müller glial cells and bipolar cells, the main second-order cells of the retina, in the healthy rat retina. The topic of the second part of the thesis is the evaluation of gliotic alterations of Müller cells in a transgenic rat model of retinal degeneration (CMV-PKD21/703 HA rats), in order to obtain indications for a pathogenic role of reactive glial cells in the development of retinal degeneration and edema. Various methods were used including immunohistochemical stainings, real-time RT-PCR, patch-clamp recordings, and cell swelling experiments. The data suggest that both neurons and reactive Müller cells may contribute to formation of retinal edema. In contrast to Müller cells, bipolar cells are apparently not capable to regulate the extracellular space volume in the healthy retina. However, reactive Müller cells are impaired in their capability to regulate retinal water and ion homeostasis. Impaired regulation of the extracellular space volume may result in neuronal hyperexcitation and degeneration.