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Thesis (Ph. D.)--Virginia Commonwealth University, 2006. / Prepared for: Dept. of Anatomy and Neurobiology. Bibliography: leaves 159-181. Also available online.

Traumatic brain injury (TBI) is a common event that can lead to profound consequences for the individual involved, and a considerable socio-economic cost. The initial injury event triggers a series of secondary brain injury mechanisms that lead to further mortality and contribute to morbidity. One classical injury pathology is termed traumatic axonal injury (TAI), which in clinical settings produces the picture of diffuse axonal injury. TAI occurs both as a primary insult, and as a consequence of secondary mechanisms. One secondary injury mechanism that worsens TAI may be Wallerian degeneration (WD), a cell-autonomous axonal death pathway. The relationship between traumatic axonal injury and WD is poorly characterised. This thesis explores the basic mechanisms by which a physical axonal trauma can lead to WD, and how modulation of the WD pathway can affect the cellular responses to a traumatic injury. This involves the development and characterisation of in vitro and in vivo models of traumatic axonal injury. These models are then used to explore the response of cellular cultures to injury when treated with pharmacological and genetic modulators of WD. Using a primary neuronal stretch-injury system we demonstrate that rates of neurite degeneration are altered by modulators of the WD pathway but that a purported neuroprotective compound ‘P7C3-A20’ did not protect primary cultures in vivo and did not act via a WD dependent mechanism. An organotypic hippocampal slice stretch injury model was then used to demonstrate genetic rescue of cellular death, and used to assess amyloidogenic responses to injury. Next we established a TBI model using Drosophila Melanogaster, and demonstrated that a loss of function mutation in a key WD gene ‘highwire’ which controls NMNAT levels, was capable of rescuing premature death and a range of behavioral deficits after a high impact trauma. The injury caused dopaminergic neuronal loss and this was rescued by highwire mutation. Furthermore, this dopaminergic neuronal protection extended to a genetic PINK1 model of Parkinsonism. Together these results help establish WD as an important secondary injury mechanism in TBI, and provide evidence that modulation of the WD pathways can improve outcomes in various model systems. This provides a foundation for future translational research into the fields of WD and TBI.

Neurons establish complex networks within the nervous system allowing for rapid cell-cell communication via their long, thin axonal processes. These wire-thin projections are susceptible to a number of insults or injuries, and axonal damage can lead to disruption in signal propagation and an overall dysfunction of the neural network. Recent research focused on investigating the underlying mechanisms of injury-induced axon degeneration led to the discovery of a number of endogenous, pro-degenerative molecules such as dSarm/Sarm1, Highwire/Phr1, and Axundead. These signaling molecules are thought to execute axon degeneration in response to injury locally within the distal severed axon, but the exact mechanism of action is unclear. To further identify novel participants of the axon death signaling cascade, we performed an unbiased forward genetic mutagenesis screen using the sensory neurons within the adult wing of Drosophila melanogaster. We identified a novel role for the C2H2 zinc finger transcription factor, Pebbled (Peb)/Ras-responsive element binding protein 1 (RREB1) in partially suppressing injury-induced axon degeneration. Loss of function peb mutant glutamatergic neurons present two distinct axon degeneration defects: either complete protection from axotomy, or they exhibit a novel phenotype in which axons fragment into long, continuous pieces instead of undergoing complete degeneration. Additionally, we show an enhancement of the peb protective phenotype when dSarm levels are decreased, but not with reduced levels of axundead. These data provide the first evidence of a transcription factor involved in regulating injury-induced axon degeneration signaling in vivo.

Neurons comprise the main information processing cells of the nervous system. To integrate and transmit information, neurons elaborate dendritic structures to receive input and axons to relay that information to other cells. Due to their intricate structures, dendrites and axons are susceptible to damage whether by physical means or via disease mechanisms. Studying responses to axon injury, called Wallerian degeneration, in the neuronal processes of Drosophila melanogaster has allowed the identification of genes that are required for injury responses. Screens in Drosophila have identified dsarm and highwire as two genes required for axon degeneration; when these genes are mutated axons fail to degenerate after injury, even when completely cut off from the neuronal cell body. We found that these genes are also required for dendrite degeneration after injury in vivo. Further, we reveal differences between axon and dendrite injury responses using in vivo timelapse recordings and GCaMP indicators of intracellular and mitochondrial calcium transients. These data provide insights into the neuronal responses to injury, and better define novel targets for the treatment of neurodegenerative diseases.

The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from PDF of title page (University of Missouri--Columbia, viewed on March 25, 2010). Vita. Thesis advisor: Christian Lorson. "July 2009" Includes bibliographical references.

Submitted by Cássia Santos (cassia.bcufg@gmail.com) on 2016-06-16T11:54:04Z No. of bitstreams: 2 Tese - Camila Franca de Paula Orlando - 2015.pdf: 2619656 bytes, checksum: cb74bf2b84fcf86e2eb757ba7b9413a2 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-06-28T13:10:17Z (GMT) No. of bitstreams: 2 Tese - Camila Franca de Paula Orlando - 2015.pdf: 2619656 bytes, checksum: cb74bf2b84fcf86e2eb757ba7b9413a2 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2016-06-28T13:10:17Z (GMT). No. of bitstreams: 2 Tese - Camila Franca de Paula Orlando - 2015.pdf: 2619656 bytes, checksum: cb74bf2b84fcf86e2eb757ba7b9413a2 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2015-12-21 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / This thesis presents two different studies about neurotoxicity in laboratory animals. The first study was about a sintetic analgesic called tramadol hydrochloride. The second study is about an natural poison plant called Cicuta douglasii. Tramadol is considered a safe drug as compared to other opioids. However, it has been suggested that spinal administration may trigger neurotoxicity. The objective of this study was to evaluate the neurotoxic effects of tramadol administered by spinal injection in rabbits. Thirty-two rabbits were randomly divided into two groups of 16 animals, with a control group (CG) and a tramadol group (TG). Each group was divided into four subgroups according to the application site (epidural and intrathecal) and evaluation time (seven and 30 days). For administration of tramadol or saline into the epidural or intrathecal space, a polyethylene catheter was implanted between the sixth and seventh lumbar spinous process where it remained for seven consecutive days in all animals. Various clinical and histology evaluations were conducted including determining the treatment effects on the frequency and severity of vacuolization, gliosis, inflammatory infiltrate, heart attacks, bleeding, chromatolysis, cellular edema, Wallerian degeneration, and malacia. Significant differences were found between groups for the Wallerian degeneration. Wallerian degeneration was more frequent in TG than in the CG. We conclude that tramadol can worsen nerve fiber lesions, as shown by Wallerian degeneration caused by catheterization of the epidural and intrathecal space in rabbits. Water hemlock are plants of the genus Cicuta and are toxic to animals and humans. The primary toxin is, cicutoxin, which is present in large concentrations in the tubers; other parts of the plant are toxic, but may contain lower concentrations of cicutoxin. Further, other forms of cicutoxin such as cicutols may contribute to toxicity in various plant parts. The objective of this study was to determine the toxicity of different parts of Cicuta douglasii and characterize their behavioral effects in mice. An aqueous extract was made of green seeds, dry seeds, tubers, flowers and stems of Cicuta douglasii and dosed orally to mice to determine the LD50 (the up and down test). The results indicated that only the green seeds and tubers were toxic to animals by inducing clonic-tonic seizures and death. The LD50 was 17mg/kg and 1320mg/kg for tubers and green seed, respectively. Several tests were used to evaluate motor function and behavior in treated vs. control mice including Grip Strength, Rotarod, Tremor Monitor and Open Field. For each test 12 animals were used per group. The control group (CG) received saline orally, one group received 40% of the LD50 orally (G40) and another group received 85% of the LD50 (G85). The animals were evaluated before gavage and 30 min, 90 min, 120 min, 150 min, 180 min, 240 min and 300 min after gavage. In summary, Cicuta douglasii affected muscle function of mice, including their ability to grasp and hold onto objects, their balance and motility on a Rotarod, motor activity, and exploratory and anxiety-related (i.e., thigmotaxis) behaviors. Seizures interspersed with CNS depression were observed in animals poisoned by Cicuta douglasii. / Esta tese apresentou dois distintos estudos sobre neurotoxicidade em animais de laboratório. O primeiro estudo envolveu um analgésico sintético denominado cloridrato de tramadol. O segundo estudo envolveu uma planta tóxica denominada Cicuta douglasii e também conhecida como Water Hemlock. O tramadol é considerado um fármaco seguro se comparado aos outros opioides. No entanto, foi sugerido que se administrado por via espinhal pode desencadear sinais de neurotoxicidade. Baseando-se nesta afirmativa foi proposto com este estudo avaliar os efeitos neurotóxicos do tramadol aplicado por via espinhal em coelhos. Foram utilizados para o estudo 32 coelhos aleatoriamente divididos em dois grupos de 16 animais, grupo controle (GC) e grupo tramadol (GT). Cada grupo foi subdividido em mais quatro subgrupos de acordo com o local de aplicação (epidural e intratecal) e o tempo de avaliação (sete e 30 dias). Para administração do tramadol ou da solução salina nos espaços epidural ou intratecal foi realizado a implantação de um cateter de polietileno entre o sexto e o sétimo processo espinhoso lombar onde permaneceu durante sete dias consecutivos em todos os animais. Para avaliação histológica, as variáveis analisadas foram presença de vacuolização, gliose, infiltrado inflamatório, infarto, hemorragia, cromatólise, edema celular, degeneração walleriana e malácia. Foram encontradas diferenças significativas entre os grupos na variável degeneração walleriana, no qual se verificou uma maior frequência no GT que no GC. Conclui-se que o tramadol pode agravar as lesões, representadas por degeneração walleriana, provocadas pela cateterização crônica do espaço epidural e intratecal em coelhos. As water hemlocks são plantas do gênero Cicuta e consideradas tóxicas para animais e humanos. Sua principal toxina conhecida, a cicutoxina, está presente em grandes concentrações no tubérculo da planta, contudo, estudos mais recentes têm sugerido que outras partes da planta são tóxicas. Face ao exposto, este estudo teve como finalidade determinar a toxicidade de diferentes partes da Cicuta douglasii e caracterizar seus efeitos comportamentais em camundongos. Foi utilizado um extrato aquoso de sementes verdes, sementes secas, tubérculos, flores e caules de Cicuta douglasii e administrado via oral em camundongos para determinação da DL50 (teste de up and down). Os resultados indicaram que apenas o tubérculo e as sementes verdes são tóxicos para os animais induzindo convulsões tônico-clônicas e morte e as respectivas DL50 foram 17mg/kg e 1320 mg/kg. Para os testes de função motora e de comportamento foram utilizados o GripStrenght, Rota Rod, Tremor Monitor e Campo Aberto. A dose utilizada para os testes foi 40% (10 ml/kg) e 85% (20 ml/kg) da DL50. Para cada teste foram utilizados 12 animais por grupo. O grupo controle (GC) recebeu solução salina por via oral, um grupo recebeu 40% da DL50 por via oral (G40) e outro grupo recebeu 85% da DL50 (G85). Os animais foram avaliados antes da gavagem e 30min, 90 min, 120 min, 150 min, 180 min, 240 min e 300 min após a gavagem. De modo geral, pode-se concluir que a Cicuta douglasii afetou a função muscular de camundongos, a habilidade de preensão dos animais a objetos, a coordenação motora, a atividade motora e o comportamento. Crises convulsivas intercaladas com depressão do SNC foram observadas nos animais intoxicados pela Cicuta douglasii

A significant component of diffuse brain injury (DBI) is diffuse axonal injury (DAI) which is responsible for the morbidity and mortality associated with this condition. DAI and its experimental counterpart traumatic axonal injury (TAI) result in scattered microscopic pathology characterized by focal impairment of axonal transport leading to progressive swelling and delayed axotomy. DBI-mediated perisomatic axotomy does not result in acute neuronal death suggesting that delayed axotomy was responsible for this unanticipated response. To evaluate this hypothesis, we examined the spatiotemporal progression of DBI-mediated perisomatic TAI. LM / TEM identified impaired axonal transport within 15 - 30 min post-injury. Perisomatic TAI revealed somata and related proximal / distal axonal segments with normal ultrastructural detail continuous with axonal swellings. In other cases, axotomy was confirmed by loss of axonal continuity distal to the swelling. By 60 - 180 min post-injury, somatic, proximal segment, and swelling ultrastructure were comparable to earlier time points although swelling diameter increased. Distal segment ultrastructure revealed the initial stages of Wallerian degeneration. Axotomy sites did not internalize pre-injury administered dextran suggesting pathogenesis independent of altered axolemmal permeability. Given the rapidity of perisomatic axotomy, absence of axolemmal permeability may constitute the more significant finding in terms of somatic protection.DBI-mediated neuroinflammatory reactions were then examined to see if this non-lethal neuronal pathology evoked responses comparable to those following focal injury. Microglia / macrophage responses within diffusely injured loci uncomplicated by focal pathology were explored using LM, TEM, and confocal evaluations as was albumin immunoreactivity to assess injury-induced blood-brain barrier (BBB) alterations. Initially, microglial activation was observed within injured loci while microglia within adjoining regions maintained resting phenotypes. Scattered activated microglia were observed among injured axons though no clear associations were seen. Later, activated microglia contained myelin debris while only limited microglial aggregations were recognized. Macrophages also localized to injured loci with select cells approximating somata of axotomized neurons. Immune cell observations correlated with altered BBB permeability. These data indicated rapid, yet initially uncoordinated, and persistent immune cell reactivity to DBI pathology. Taken together, these responses suggest that histopathological evaluation following DBI may include non-lethal neuronal injury with unique neuroinflammatory findings.

Introdução: A simpatectomia torácica é o único tratamento, definitivo e eficaz, para a hiperidrose primária. A ramicotomia é um procedimento cirúrgico tão eficaz, mais conservador e com menos efeitos adversos que a simpatectomia convencional, contudo foi abandonada pela alta taxa de recidiva, atribuída, até então, à secção incompleta dos ramos comunicantes, ao desenvolvimento de outras vias de condução para o estímulo central e à regeneração neural. A avaliação histológica dos ramos comunicantes simpáticos após a ramicotomia, pode ajudar a entender o processo de recidiva dos sintomas da hiperidrose e, dessa forma contribuir para o desenvolvimento de estratégias para evitá-la. MATERIAL E MÉTODOS: 28 suínos foram submetidos à ramicotomia por videotoracospia e divididos randomicamente em 5 grupos, sacrificados com 15, 45, 90, 135 e 180 dias de pós-operatório (DPO). Os segmentos operados foram removidos cirurgicamente e submetidos à avaliação macroscópica da regeneração assim como análise histológica dos ramos comunicantes brancos e cinzentos para quantificação da reação inflamatória, deposição de fibras de colágeno grossas e finas, fibras reticulares e células de Schwann por imuno-histoquímica. Os dados foram comparados ao grupo controle, composto por segmentos intactos, não operados. RESULTADOS: Não houve regeneração macroscópica no grupo de 15 DPO sendo presente em 41,6% dos casos no grupo 180 DPO (p < 0,05). A reação inflamatória foi determinante no processo de degeneração Walleriana, com presença importante das células de Schwann nos ramos pré-ganglionares (p < 0,05), as células de Schwann apresentaram evolução semelhante nos dois ramos a partir do grupo de 45DPO, mantendo-se em menor número nos ramos cinzentos. As fibras de colágeno foram cruciais na cicatrização e as fibras reticulares importantes na regeneração neural, com correlação negativa entre elas (r = - 0,414; p < 0,01). A deposição de fibras de colágeno foi maior nos ramos cinzentos, apresentando pico de deposição no grupo 135 DPO e declínio importante no grupo 180 DPO (p < 0,05). CONCLUSÕES: A ramicotomia permite a secção completa de todos os ramos comunicantes do gânglio simpático. A taxa de regeneração histológica deve ser maior que a taxa de recidiva dos sintomas no humano, devido a regenerações não funcionais. O processo regenerativo é similar nos ramos brancos e cinzentos, com tendências menores para os últimos. A regeneração dos ramos comunicantes deve ser um dos principais fatores de recidiva da hiperidrose após a ramicotomia / INTRODUCTION: Thoracic sympathectomy is the only definitive and efficient treatment for primary hyperhidrosis. The ramicotomy is a surgical procedure that is as efficient as conventional sympathectomy but more conservative, having less adverse effects then conventional sympathectomy. This procedure was abandoned on account of the high recurrence rate, attributed to the incomplete section of the rami communicantes and to the development of new pathways of conduction to the central stimuli. MATHERIAL AND METHODS: Twenty-eight swine underwent bilateral videothoracoscopic ramicotomy and were randomly divided into 5 groups. The animals were sacrificed at 15, 45, 90, 135 and 180 days post-operative POD. The segments were removed and evaluated for macroscopic regeneration and histological analysis of the white and gray rami communicantes analyzing the inflammatory reaction, deposition of thin and thick collagen fibers, reticular fibers and Schwann cells. The data was compared to intact segments of sympathetic trunk as a positive control. RESULTS: There was neither macroscopic nor microscopic regeneration at the 15 POD group. The remaining groups had an average of 41,6% of regeneration, more significant at the 180 POD group (p<0.05). The inflammatory reaction was crucial in the process of Wallerian degeneration, with an important participation of the Schwann cells in the pre-ganglionic rami (p<0.05). The Schwann cells presented a similar evolution in both rami beginning at the 45 POD group, with a smaller count in the gray rami. The collagen fibers were significant in the cicatrization and the reticular fibers were important in neural regeneration, with a meaningful negative correlation between them (r = - 0,414; p < 0,01). The rate of deposition of the collagen fibers was greater in the white rami when compared to the gray rami in the first trimester and less important in the second trimester (p<0.05). CONCLUSIONS: Ramicotomy allows complete section of all rami communicantes of the sympathetic ganglia. The histological regeneration might be greater than the recurrence rates of clinical symptoms as seen in the human being due to non-functional regenerations. The restoration process is similar in both white and gray rami, with smaller tendencies in the last one. The regeneration of the could be one of the main factors for recurrence of hyperhidrosis following ramicotomy

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