<p>The goal of this study is to use a rat model of acquired temporal lobe epilepsy, pilocarpine-induced status epilepticus (SE), to test the hypothesis that WD exacerbates the inflammatory consequences of SE in hippocampus of male rats. Testing this hypothesis in animals consuming WD from early in life may provide insight into how a modifiable environmental factor could influence the development of seizure disorders. </p>

Microglia activity has been detected in Parkinson’s disease (PD) post-mortem brains and experimental animal models; however the precise interplay between microglia and dopamine neurons of the SNpc is not well understood. In the blood plasma of PD patients, our laboratory found elevated levels of interferon-gamma (IFN-γ), a proinflammatory cytokine and potent activator of microglia. Given this, we sought to untangle the immune responses relevant to PD in mice, examining IFN-γ’s involvement and signaling mechanism using an inflammatory co-culture model of microglia and midbrain neurons treated with rotenone. By means of RT-PCR, we discovered IFN-γ mRNA transcripts are produced by microglia, and this expression increases upon exposure to rotenone. We delineated IFN-γ’s signaling mechanism in co-cultures using different IFN-γ receptor deficient cells, and showed it engages receptors in an autocrine (not paracrine) manner to further microgliosis and dopamine cell loss. After exploring the innate immune response in a model of PD, we subsequently shifted focus to an in vivo system to better investigate any involvement of the delayed humoral arm of the adaptive immune system. Needing a time appropriate death paradigm, we developed a protracted low dose regimen of MPTP, which elicits dopaminergic cell death after 2 weeks of treatment. Subjected to this paradigm, Rag 2 mutant mice (deficient in both T and B cells) exhibit resistance to dopamine cell loss, microglia activation and motor impairments. Further evidence in support of immune involvement came with the resensitization of Rag2 mice to MPTP after reconstitution with WT splenocytes. Additionally, mice deficient in Fcγ receptors exhibited neuroprotection in our protracted degeneration model. Taken together, these data indicate the innate and humoral arm can modulate the microglial response to dopaminergic degeneration and may participate in Parkinson's disease.

Microglia activity has been detected in Parkinson’s disease (PD) post-mortem brains and experimental animal models; however the precise interplay between microglia and dopamine neurons of the SNpc is not well understood. In the blood plasma of PD patients, our laboratory found elevated levels of interferon-gamma (IFN-γ), a proinflammatory cytokine and potent activator of microglia. Given this, we sought to untangle the immune responses relevant to PD in mice, examining IFN-γ’s involvement and signaling mechanism using an inflammatory co-culture model of microglia and midbrain neurons treated with rotenone. By means of RT-PCR, we discovered IFN-γ mRNA transcripts are produced by microglia, and this expression increases upon exposure to rotenone. We delineated IFN-γ’s signaling mechanism in co-cultures using different IFN-γ receptor deficient cells, and showed it engages receptors in an autocrine (not paracrine) manner to further microgliosis and dopamine cell loss. After exploring the innate immune response in a model of PD, we subsequently shifted focus to an in vivo system to better investigate any involvement of the delayed humoral arm of the adaptive immune system. Needing a time appropriate death paradigm, we developed a protracted low dose regimen of MPTP, which elicits dopaminergic cell death after 2 weeks of treatment. Subjected to this paradigm, Rag 2 mutant mice (deficient in both T and B cells) exhibit resistance to dopamine cell loss, microglia activation and motor impairments. Further evidence in support of immune involvement came with the resensitization of Rag2 mice to MPTP after reconstitution with WT splenocytes. Additionally, mice deficient in Fcγ receptors exhibited neuroprotection in our protracted degeneration model. Taken together, these data indicate the innate and humoral arm can modulate the microglial response to dopaminergic degeneration and may participate in Parkinson's disease.

No description available.

Lysosomal storage disorders are a group of rare neurodegenerative diseases that are collectively common, sharing many aspects with other neurodegenerative disorders, including substrate build-up and neuroinflammation. The GM2 Gangliosidoses, Tay-Sachs disease and Sandhoff disease, are pathologically overlapping lysosomal storage disorders, with high prevalence within specific ethnicities. Their effects are neurologically devastating and often fatal at young ages. Current treatments only slow or stall an inevitable decline in health. Novel treatment targets are needed for these disorders, and others with similar pathologies. In these works we demonstrate the negative effect the inflammatory cytokine tumour necrosis factor-alpha has on survival of a model of Sandhoff disease. We demonstrate its role in the upregulation of astrogliosis, and apoptosis, and we present evidence that this effect on astrogliosis occurs through an upregulation of the JAK-2/STAT3 pathway. Though fruitful, a singular focus on inflammation/gliosis in these diseases has left a vacuum in the research into neuron specific molecular processes. We observe the development of inflammation, astrogliosis and neuronal processes in our model, and demonstrate a bi-phasic disease progression, in which early onset microgliosis precedes terminal astrogliosis, apoptosis, and a decline in excitatory glutamate receptors, suggesting neuron-specific malfunction. Furthermore, we show that knockout of the synaptic protein neuronal pentraxin 1 retards neurodegeneration and extends the lifespan of Sandhoff disease mice, independent of inflammation or astrogliosis. Through electrophysiology, we provide evidence of dysregulation of glutamate receptors in Sandhoff disease, and show that knockout of neuronal pentraxin 1 provides rescue from this dysregulation. This work expands on research into gliosis in GM2 gangliosidoses, presents the finding of a novel protein isoform, and presents a new focus on non-glial disease mechanisms and treatments for these and other neurodegenerative disorders. / Thesis / Doctor of Philosophy (PhD) / Lysosomal storage disorders are a group of neurological diseases that are debilitating, and often fatal at a young age. Two diseases of this group- Tay-Sachs disease and Sandhoff disease – are similar in their causes and symptoms. Current treatments for these diseases only slow or stall an inevitable decline in health. New targets for treatment are required, and we provide data suggesting several proteins that may fit this criterion. We also provide evidence of the discovery of a new form of one of these proteins, which is found in high levels in the disease, indicating it may be important in these and other neurodegenerative diseases. Finally, we provide findings indicating that a certain cell type, which is largely ignored in current research for these diseases, may be important in the disease progress. These findings increase our knowledge of Tay-Sachs disease and Sandhoff disease, and open new avenues for medicinal intervention.

Chronic pain is a continuous or recurring pain, which exceeds the normal course of recovery to an injury or disease. According to the chronic pain origin it can be classified as inflammatory or neuropathic. The neuropathic pain is a type of chronic pain, characterized by abnormalities such as dysesthesia, hyperalgesia and allodynia, interfering the patient's quality of life and is difficult to treatment. In view of this, and considering that (-)-α-bisabolol (BIS) presents several promising pharmacological properties for the relief of this type of pain, this study aimed to evaluate the antinociceptive and anti-inflammatory effect of pure BIS and its complex of inclusion in β-cyclodextrin (β-CD) in preclinical models of chronic pain. Thus, the complexing efficiency of BIS and β-CD was investigated by means of High Performance Liquid Chromatography (HPLC). The Chronic pain models were induced by injection of CFA (25 μl; i.pl.) and partial lesion of the sciatic nerve (PNSL). The animals were evaluated for behavioral parameters: mechanical hyperalgesia, thermal hyperalgesia, muscle strength and motor coordination. In addition, we assessed the concentrations the cytokines of TNF-α and IL-10, the expression of the ionized calcium-binding adapter protein (IBA-1). Animals were treated with BIS and BIS/β-CD (50 mg/kg, p.o) or vehicle. The complexation efficiency (EE%) of BIS in inclusion complexes with β-CD is equal to 50% ± 0.19. From this result, it is inferred that the amount of BIS included in the β-CD cavity is less than 50 mg/kg. Then, by means of HPLC analysis and calculation of dose concentration, it was possible to determine that the amount of BIS in β-CD was about 5 mg/kg. Despite this difference in doses between BIS and BIS/β-CD, a significant reduction (p <0.001) in mechanical hyperalgesia was observed in both pain models studied, and a significant reduction (p <0.001) in thermal hyperalgesia in the model of DN. No alterations were found in muscle strength and motor coordination. In addition, both compounds inhibit (p <0.05) TNF-α production in the sciatic nerve and spinal cord and stimulate (p <0.05) the release of IL-10 in the spinal cord in PNSL induced-mice. Further, BIS and BIS/β-CD reduce IBA-1 immunostaining compared to vehicle treated-mice. Therefore, BIS and BIS/β-CD attenuates the hyperalgesia, misregulated the cytokines release and inhibit IBA-1expression in spinal cord in PNSL model. Therefore, the results show that BIS and BIS/β-CD have an antihyperalgesic effect in chronic inflammatory pain model and DN model and that this effect may be associated to the modulation of pro and anti-inflammatory cytokines and inhibition of microgliosis, which leads to decreased release of cytokines and hypersensitivity to noxious stimuli. These facts are currently considered as important therapeutic strategies for the treatment of DN. Thus, we can suggest that BIS and BIS/β-CD are promising molecules for the treatment of chronic pain such as neuropathic pain. / A dor crônica é uma dor contínua ou recorrente, que excede o curso normal de recuperação para uma lesão ou doença. A dor neuropática (DN) é um tipo de dor crônica, caracterizada por anormalidades como disestesia, hiperalgesia e alodinia, interfere negativamente na qualidade de vida do paciente e é de difícil tratamento. Diante disto, e considerando que (-)-α-bisabolol (BIS) apresenta diversas propriedades farmacológicas promissoras para o alívio desse tipo de dor, este estudo teve como objetivo avaliar o efeito antinociceptivo e anti-inflamatório do BIS puro e do seu complexo de inclusão em β-ciclodextrina (β-CD) em modelos pré-clínicos de dor crônica. Assim, investigou-se a eficiência de complexação do BIS e β-CD por meio de Cromatografia Líquida de Alto Desempenho (HPLC). Os modelos de dor crônica foram induzidos por injeção de CFA (25 μl; i.pl.) e lesão parcial do nervo ciático (LPNC). Os animais foram avaliados quanto aos parâmetros comportamentais: hiperalgesia mecânica, hiperalgesia térmica, força muscular e coordenação motora. Avaliaram-se também as concentrações das citocinas TNF-α e IL-10, a expressão da proteína adaptadora de ligação de cálcio ionizado (IBA-1). Os animais foram tratados com BIS e BIS/β-CD (50 mg/kg, v.o.) ou veículo. A eficiência de complexação (EC%) de BIS em complexos de inclusão com β-CD foi igual a 50% ± 0,19. A partir deste resultado, infere-se que a quantidade de BIS incluído na cavidade da β-CD é inferior a 50 mg/kg. Então, por meio da análise em HPLC e cálculo de concentração de doses, foi possível determinar que a quantidade de BIS em β-CD foi cerca de 5 mg/kg. Apesar desta diferença de doses entre BIS e BIS/β-CD, em ambos foi observado uma redução significativa (p<0,001) na hiperalgesia mecânica, nos dois modelos de dor estudados, e redução significativa (p <0,001) na hiperalgesia térmica no modelo de DN. Não foram encontradas alterações na força muscular e nem na coordenação motora. Além disso, ambos os compostos inibem (p <0,05) a produção de TNF-α no nervo ciático e na medula espinhal e estimulam (p <0,05) a liberação de IL-10 na medula espinhal em camundongos induzidos com LPNC. E que BIS e BIS/β-CD reduzem a imunomarcação IBA-1 em comparação com os camundongos tratados com veículo. Portanto, BIS e BIS/β-CD atenuam a hiperalgesia, modulam a liberação de citocinas e inibem a expressão de IBA-1 na medula espinhal no modelo LPNC. Com isso, os resultados mostram que BIS e BIS/β-CD possuem efeito anti-hiperalgésico em modelo de dor inflamatória crônica e em modelo de DN e que este efeito pode estar associado à modulação de citocinas pró e anti-inflamatórias e inibição da microgliose, que gera diminuição da liberação de citocinas e da hipersensibilidade ao estímulo nocivo. Fatos estes considerados na atualidade como estratégias terapêuticas importantes para o tratamento de DN. Assim, podemos sugerir que o BIS e BIS/β-CD, são moléculas promissoras para o tratamento de dores crônicas como a neuropática. / Aracaju