Submitted by Ramon Santana (ramon.souza@ufpe.br) on 2015-03-13T19:49:52Z No. of bitstreams: 2 DISSERTAÇÃO Giselle Machado M. Moreno .pdf: 1689109 bytes, checksum: 2d05742bbd9d111da0b3253b85d9846c (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) / Made available in DSpace on 2015-03-13T19:49:53Z (GMT). No. of bitstreams: 2 DISSERTAÇÃO Giselle Machado M. Moreno .pdf: 1689109 bytes, checksum: 2d05742bbd9d111da0b3253b85d9846c (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Previous issue date: 2014-03-07 / Conselho Nacional de Desenvolvimento Cientìfico e Tecnológico – CNPq / A estimulação transcraniana por corrente contínua (ETCC) consiste na aplicação de corrente direta de baixa intensidade através do crânio e tem se mostrado eficaz no tratamento de diversas desordens neurológicas e psiquiátricas. Dentre as técnicas de estimulação do sistema nervoso central a ETCC ocupa posição de destaque por ser capaz de modular a excitabilidade cortical com vantagens como: não ser invasiva, ser indolor, de baixo custo, fácil uso e fácil mascaramento na realização de estudos. No entanto, apesar de extensas pesquisas sobre os efeitos da ETCC em diversos estados patológicos, seus mecanismos básicos de ação permanecem desconhecidos. Partindo do entendimento do grande envolvimento glial e de moléculas envolvidas no crescimento axonal na dinâmica de funcionamento das sinapses e excitabilidade cortical, o objetivo deste estudo foi verificar os efeitos da ETCC anódica sobre parâmetros celulares e moleculares relacionados à plasticidade sináptica. Foram utilizados 20 ratos Wistar machos adultos, divididos aleatoriamente em dois grupos: (i) ETCC ativa anódica (E), e (ii) ETCC fictícia, sham (S). Os animais receberam ETCC anódica com intensidade de corrente igual a 400 μA, durante 10 minutos por dia, durante cinco dias consecutivos. Após o tratamento foi feita análise imunohistoquímica para reatividade microglial (Iba1) e astrocitária (GFAP), foram investigadas possíveis alterações teciduais estruturais (HE) e degeneração neuronal (FJC), bem como quantificação da expressão da proteína associada ao crescimento axonal, GAP-43. Os ratos do grupo E apresentaram aumento de ~90% na expressão da proteína GAP-43 em homogenados de todo o córtex cerebral (p = 0.032) e na reatividade microglial por uma extensa área cortical em torno da região estimulada, quando comparados ao grupo S. Não foram observadas alterações anatomopatológicas no tecido nem sinais de astrogliose ou neurodegeneração no córtex cerebral dos animais que receberam ETCC. Conclui-se que os parâmetros de estimulação utilizados no presente estudo são capazes de induzir alterações moleculares e celulares no córtex cerebral de animais saudáveis, na ausência de injúria ao tecido nervoso. É possível que tais efeitos estejam envolvidos em algumas das ações da ETCC sobre a plasticidade sináptica e excitabilidade cortical.

No description available.

The regenerative capacity of mature mammalian CNS neurons after axonal injury is severely limited by a variety of mechanisms. Retrograde repression is the continuous inhibition of the expression of growth phenotypes by tonic signals produced by target tissues and transmitted to the neuron cell body via retrograde axonal transport. Loss of target contact through axonal injury is thought to interrupt this retrograde signal and allow the up-regulation of growth-associated proteins. Most CNS neurons, however, possess many widespread axon collaterals, such that retrograde repression is maintained by intact sustaining collaterals even if some axons are injured.<p>In this project we investigated whether or not retrograde repression plays a role in limiting the expression of GAP-43 in transcallosal neurons. Because TCNs possess local axon collaterals to nearby cortex and project distal axons to homologous areas of contralateral cortex, we hypothesized that the simultaneous interruption of retrograde repressive signals from both ipsilateral and contralateral cortex would result in an up-regulation of GAP-43 expression in at least some TCNs.<p>We found that a bilateral infusion of a function blocking antibody to FGF-2 into the parietal cortex of rats using implanted osmotic mini-pumps resulted in a significant increase in the level of expression of GAP-43 mRNA in TCNs identified by retrograde fluorescent labeling. In contrast, neither ipsilateral or contralateral antibody infusions alone increased GAP-43 expression significantly compared to controls. The level of expression of GAP-43 in TCNs did not significantly increase after stereotactic callosotomy alone, but callosotomized animals treated with an ipsilateral infusion of anti-FGF-2 had levels of increased GAP-43 expression equivalent to those seen in animals that had received bilateral antibody infusions.<p>We conclude that FGF-2 provides a retrograde repressive signal for at least some mature mammalian TCNs, and that the expression of growth-associated proteins can be up-regulated in CNS neurons by simultaneously blocking retrograde repressive signals from all existing axon collaterals. The ability to alter the gene expression of mature CNS neurons in both normal and injured states through the targeted infusion of a pharmacological agent may have potential clinical implications in the future.

The regenerative capacity of mature mammalian CNS neurons after axonal injury is severely limited by a variety of mechanisms. Retrograde repression is the continuous inhibition of the expression of growth phenotypes by tonic signals produced by target tissues and transmitted to the neuron cell body via retrograde axonal transport. Loss of target contact through axonal injury is thought to interrupt this retrograde signal and allow the up-regulation of growth-associated proteins. Most CNS neurons, however, possess many widespread axon collaterals, such that retrograde repression is maintained by intact sustaining collaterals even if some axons are injured.<p>In this project we investigated whether or not retrograde repression plays a role in limiting the expression of GAP-43 in transcallosal neurons. Because TCNs possess local axon collaterals to nearby cortex and project distal axons to homologous areas of contralateral cortex, we hypothesized that the simultaneous interruption of retrograde repressive signals from both ipsilateral and contralateral cortex would result in an up-regulation of GAP-43 expression in at least some TCNs.<p>We found that a bilateral infusion of a function blocking antibody to FGF-2 into the parietal cortex of rats using implanted osmotic mini-pumps resulted in a significant increase in the level of expression of GAP-43 mRNA in TCNs identified by retrograde fluorescent labeling. In contrast, neither ipsilateral or contralateral antibody infusions alone increased GAP-43 expression significantly compared to controls. The level of expression of GAP-43 in TCNs did not significantly increase after stereotactic callosotomy alone, but callosotomized animals treated with an ipsilateral infusion of anti-FGF-2 had levels of increased GAP-43 expression equivalent to those seen in animals that had received bilateral antibody infusions.<p>We conclude that FGF-2 provides a retrograde repressive signal for at least some mature mammalian TCNs, and that the expression of growth-associated proteins can be up-regulated in CNS neurons by simultaneously blocking retrograde repressive signals from all existing axon collaterals. The ability to alter the gene expression of mature CNS neurons in both normal and injured states through the targeted infusion of a pharmacological agent may have potential clinical implications in the future.

Successful axon regeneration appears to depend on the development of an injury response. Dorsal root ganglion neurons exemplify the necessity of this injury response in a unique way. Peripheral nerve transection leads to development of an injury response and successful regeneration whereas central root transection does neither. The injury response may involve extracellular and intracellular pathways. To investigate the extraneuronal influences, we performed nerve transection of either the central or peripheral axon branches and studied the expression of GAP-43, a key growth associated protein, and the transcription factors ATF3, c-Jun, and STAT3. Our results show that the responses to peripheral versus central nerve transection are fundamentally different. Peripheral but not central nerve transection increases GAP-43, ATF3, and c-Jun expression. STAT3, however, is upregulated as a result of central but not peripheral nerve transection. To investigate potential intracellular signalling pathways, we applied FGF-2, an extracellular mitogen, or an analog of cAMP, an intracellular second messenger to the cut end of the peripheral axon. Our results indicate that FGF-2 and cAMP act as activators of GAP-43 expression. On the other hand, FGF-2 and cAMP act to downregulate the expression of ATF3. FGF-2 upregulates c-Jun and the activated form of STAT3. Paradoxically, the regulation of GAP-43 expression by cAMP or by FGF-2 in vivo shows opposing results from the previously reported in vitro studies. Our present results suggest that the peripheral nerve injury response may be governed by at least three different signalling pathways.

Successful axon regeneration appears to depend on the development of an injury response. Dorsal root ganglion neurons exemplify the necessity of this injury response in a unique way. Peripheral nerve transection leads to development of an injury response and successful regeneration whereas central root transection does neither. The injury response may involve extracellular and intracellular pathways. To investigate the extraneuronal influences, we performed nerve transection of either the central or peripheral axon branches and studied the expression of GAP-43, a key growth associated protein, and the transcription factors ATF3, c-Jun, and STAT3. Our results show that the responses to peripheral versus central nerve transection are fundamentally different. Peripheral but not central nerve transection increases GAP-43, ATF3, and c-Jun expression. STAT3, however, is upregulated as a result of central but not peripheral nerve transection. To investigate potential intracellular signalling pathways, we applied FGF-2, an extracellular mitogen, or an analog of cAMP, an intracellular second messenger to the cut end of the peripheral axon. Our results indicate that FGF-2 and cAMP act as activators of GAP-43 expression. On the other hand, FGF-2 and cAMP act to downregulate the expression of ATF3. FGF-2 upregulates c-Jun and the activated form of STAT3. Paradoxically, the regulation of GAP-43 expression by cAMP or by FGF-2 in vivo shows opposing results from the previously reported in vitro studies. Our present results suggest that the peripheral nerve injury response may be governed by at least three different signalling pathways.

Acquiring knowledge of the morphological, molecular, and functional changes that occur to neurons following axotomy is a key step for a comprehensive understanding of the nervous system and how it reacts to injury. Propriospinal commissural interneurons (PCIs or CINs) are a class of neuron with axons that project through the ventral commissure to the contralateral spinal cord. My goal was to examine the morphological, molecular, and functional changes that occur to adult feline PCIs following a proximal axotomy. We first determined whether proximally axotomized PCIs develop de novo axons from their dendrites. C3 PCIs were proximally axotomized and several weeks later we stained PCIs and prepared the tissue for histological evaluation. Two primary classes of axotomized PCI were identified: those with a very short axon (called permanently axotomized) and those with an axon that projected across the injury site. Permanently axotomized PCIs had processes with morphological features typical of axons that emerged from their distal dendrites. These axonal processes of the distal dendrites also had GAP-43 (an axonal marker) and lacked MAP2a/b (a dendritic marker). We concluded that permanently axotomized PCIs develop de novo axons from distal dendrites. We then determined whether the axons that crossed the lesion site were representative of spontaneous functional regeneration. First, we showed that PCI axons regenerate through an environment that is typically highly inhibitory to regenerating axons. Second, we established that the regenerated axons conduct action potentials. Finally, we found that regenerated PCI axons form functional synaptic connections with neurons in the contralateral spinal cord. Collectively, these data indicated that spinal interneurons are capable of spontaneous functional regeneration through an injured spinal cord. PCI growth cones are complex and unlike growth cones previously described in the literature. The final study of the thesis examines the morphologies of PCI growth cones within spinal cord injury sites. We found that PCI growth cones have a wide range of morphologies that is independent of their location within the lesion site. Taken together, these data indicate that PCIs have a remarkable capacity for axonal elongation and contribute to remodelling of spinal circuitry following spinal injury. / Thesis (Ph.D, Physiology) -- Queen's University, 2009-12-07 11:21:47.036

No description available.

Traumatic brain injury (TBI) has a dramatic impact on our society in terms of mortality, morbidity, and inherently high financial costs. Formidable research efforts are being addressed to the identification of neuroprotective agents capable of ameliorating the neurological outcome after TBI. Preclinical studies have recently demonstrated lithium to be a promising neuroprotective agent for both acute ischemic brain injury and chronic neurodegenerative disease. In light of these encouraging data, we designed a lateral fluid-percussion injury (FPI) study aimed at investigating the role of early post-traumatic administration of lithium as a strategy for reducing TBI-induced motor and cognitive deficits. The optimal dose of this agent and the time window for its administration have been determined on the basis of data derived from the assessment of motor and cognitive functioning in experimental animals, as well as from the stereological quantification of neuronal survival (PID 7) within the CA3 and hilar regions of the hippocampus ipsilateral to the FPI. In addition, we attempted to elucidate the mechanisms underlying the neuroprotective properties of this drug via western blot analysis of levels of the pro-apoptotic marker caspase-3 (PID 1, 7) and two neuroplasticity markers, growth associated protein-43 (GAP-43) and brain-derived neurotrophic factor (BDNF) (PID 1, 7, 21). Our findings indicate that low-dose lithium chloride (0.125 or 0.25 mmol/kg), given either 30 min or 8 hr after lateral FPI significantly ameliorates injury-induced cognitive and motor impairment. Specifically, cell survival in the CA3 region of the hippocampus of the injured lithium-treated animals (but not in the hilus) was significantly increased compared to injured vehicle-treated animals. Western blot analyses revealed a significant increase in GAP-43 levels on PID 7 in injured animals when treated with lithium, indicating a possible mechanism for lithium-induced neuroprotection. In contrast, BDNF levels were relatively unchanged until PID 21, and caspase-3 activation was not observed at all, suggesting that these proteins play less significant roles in the observed neuroprotective effects of lithium treatment after lateral FPI. Early administration of lithium, within 8 hours after TBI, holds promise as an effective therapy to ameliorate postinjury neurobehavioral deficits and warrants further investigation in clinical TBI studies.

No description available.