Noch immer ist die Behandlung von Neuropathien mit den gängigen therapeutischen Mitteln für viele Patienten sehr unbefriedigend. Als erfolgsversprechender therapeutischer Ansatz werden zur Zeit Wege erforscht, welche direkt in die molekularen Entstehungsmechanismen pathologischer Veränderungen und regenerationsfördernder Mechanismen eingreifen, um dadurch eine Heilung von Nervenschäden zu ermöglichen. Bisher sind die Erkenntnisse über diese Mechanismen nicht vollständig genug, um daraus eine sichere Behandlungsmöglichkeit abzuleiten. Wegweisende Erkenntnisse deuten sich allerdings durch Studien von unterschiedlichen Vertretern des Zytokinnetzwerkes an - darunter auch TNF-alpha - welche als molekulare Ursache neuropathischer Veränderungen diskutiert werden. In dieser Studie wurde an Knockoutmäusen der Einfluss des jeweiligen TNF-alpha-Rezeptors auf morphologische Veränderungen nach CCI (Chronic constriction injury) und Crush-Verletzung des N. ischiadicus untersucht. Nach 3,7,15 und 36 Tagen (CCI) bzw. 3,7 und 28 Tagen (Crush) wurden in Methylenblau gefärbten Semidünnschnitten intakte und degenerierte Nervenfasern, Makrophagen, Angioproliferation, Ödembildung udn Veränderung des Anteils nicht neuronaler Zellen lichtmikroskopisch beurteilt. Zusätzlich wurden Mac-1+ Makrophagen immunzytochemisch erfasst. Die Ergebnisse zeigten in beiden Modellen und bei beiden Knockouttypen eine starke axonale Schädigung, die von einer großen endoneuroalen Makrophagenansammlung begleitet war. Bei TNF-R1-/- Mäusen war eine stärkere und verlängerte Degeneration mit entsprechend höheren Makrophagenzahlen sichtbar. In den Immunzytochemischen Färbungen wiesen die TNF-R1-/- Mäuse hingegen den geringsten Makropahgenanteil auf.Trotz der starken Schädigung war die anschließende Regeneration im Gegensatz zu WT und TNF-R2-/- Mäusen besser. Die Ödembildung war bei den TNF-R2-/- nach CCI besonders stark ausgeprägt und von einer schlechten Regeneration gefolgt. Während die gefundenen Daten auf eine Beteiligung beider Rezeptoren während degenerativer Prozesse hindeuten, scheint insbesondere TNF-R2 regenerationsfördernde Effekte zu vermitteln. / Current Treatment of neuropathic disorders is still dissatisfactory for many patients. A promising approach is the investigation of agents that directly interfere with molecular development of pathologic changes and regeneration. Up to now, consolidated findings of the underlying mechanisms are not yet sufficent to allow therapeutic intervention. Pathbreaking findings come from studies investigating different agents of the cytokine network - as e.g. TNF-alpha - that are discussed as molecular cause of neuropathic changes. This study investigated the influence of both TNF-alpha-receptors on morphologic changes after CCI (chronic constriction injury) and crush-injury of the sciatic nerve of TNF-R-knockoutmice. After 3,7,15 and 36 days (CCI), and 3,7 and 28 respectively (Crush),intact and degenerating nerve fibers, macrophages, angioproliferation, development of edema and changes in the amount of non-neuronal cells were acquired by light microscopy of toluidin-stained semithin sections. Additionally Mac-1+ macrophages were acquired via immuncytochemically stained sections. The results showed strong axonal damage in both knockout-types accompanied by large amounts of endoneurial macrophages. TNF-R1-/-mice showed a longer degeneration phase including respectively higher amounts of macrophages. In contrast the TNF-R1-/-mice revealed the fewest amount of macrophages in immunocytochemical sections. Despite the strong damage better nerve regeneration was observed compared to WT and TNF-R2-/-mice. Formation of edema was pronounced in TNF-R2-/- after CCI and followed by poorly regeneration. Whereas these findings point to a participation of both receptors in degeneration, TNF-R2 seems to support regeneration.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-06-21T11:43:47Z No. of bitstreams: 1 alexandresilvachaves.pdf: 1552343 bytes, checksum: e93b7672976058e0adc057d62686adba (MD5) / Rejected by Adriana Oliveira (adriana.oliveira@ufjf.edu.br), reason: on 2017-06-21T12:22:29Z (GMT) / Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-06-21T12:24:25Z No. of bitstreams: 1 alexandresilvachaves.pdf: 1552343 bytes, checksum: e93b7672976058e0adc057d62686adba (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-08-07T19:04:35Z (GMT) No. of bitstreams: 1 alexandresilvachaves.pdf: 1552343 bytes, checksum: e93b7672976058e0adc057d62686adba (MD5) / Made available in DSpace on 2017-08-07T19:04:35Z (GMT). No. of bitstreams: 1 alexandresilvachaves.pdf: 1552343 bytes, checksum: e93b7672976058e0adc057d62686adba (MD5) Previous issue date: 2014-08-29 / A tuberculose é uma doença infecciosa cujo principal agente etiológico é o Mycobacterium tuberculosis (Mtb). Macrófagos constituem a primeira linha de defesa contra a tuberculose, bem como o habitat preferencial para o Mtb. Antígenos específicos do Mtb tais como ESAT-6, CFP-10 e Rv1733 podem modular a produção de citocinas, influenciando o curso da infecção. Macrófagos infectados liberam uma grande quantidade de citocinas incluindo fator de necrose tumoral-alfa (TNF-a). As ações do TNF-a são iniciadas por seus dois receptores de membrana, mTNF-R1 e mTNF-R2, os quais podem sofrer clivagem proteolítica, resultando nas formas solúveis sTNF-R1 e sTNF-R2, respectivamente. No presente estudo, macrófagos RAW264.7 (2x105/mL) foram estimulados com a proteína de fusão ESAT6:CFP10 ou com o antígeno recombinante DosR, Rv1733, na concentração de 5pg/mL por 24 e 48 horas e a produção de TNF-a, bem como a expressão dos mTNF-Rs e concentração dos sTNF-Rs foram investigadas. Os sobrenadantes de cultura foram coletados e os níveis de TNF-a, IL-10, sTNF-R1 e sTNF-R2 foram avaliados por ELISA, enquanto que os níveis de óxido nítrico (NO) foram analisados pela reação de Griess. A determinação da viabilidade celular foi realizada pelo ensaio de MTT. Níveis de mTNF-R1 e mTNF-R2 foram analisados por citometria de fluxo e a expressão de iNOS e Arg-1 determinada por imunocitoquímica. Os resultados mostraram que ambos os antígenos induziram um aumento na expressão de TNF-a (p<0,05) sem afetar os níveis de NO, IL-10, iNOS e Arg-1. A expressão elevada de mTNF-R1 (p<0,05) em células RAW264.7 foi observada para ambos os antígenos após 24 horas de cultura, mas apenas o Rv1733 foi capaz de diminuir os níveis de sTNF-R1 (p<0,05) nos sobrenadantes de cultura. A expressão de mTNF-R2 e a detecção de sTNF-R2 (p<0,05) foi maior após estimulação com Rv1733, em 24 e 48 horas de cultura. Esses resultados sugerem que a proteína de fusão ESAT6:CFP10 e o antígeno DosR, Rv1733, modulam diferentemente a produção de TNF-a e a expressão de seus receptores em macrófagos, o que influenciar na suscetibilidade à infecção. / Tuberculosis is a severe infectious disease caused by Mycobacterium tuberculosis (Mtb). Macrophages provide a first line of defense against tuberculosis and major habitat for Mtb. Specific Mtb antigens, such as ESAT-6, CFP-10 and Rv1733, may modulate the production of cytokines, influencing the course of infection. Infected macrophages release a range of cytokines including tumor necrosis factor-alpha (TNF-a). TNF-a actions are triggered by its two membrane receptors, mTNF-R1 and mTNF-R2, which may undergo a shedding process, resulting in the soluble form sTNF-R1 and sTNF-R2, respectively. In the present study, RAW264.7 macrophages (2 x 105/mL) were stimulated with the fusion protein ESAT6:CFP10 or the recombinant DosR antigen Rv1733 at 5[1,g/mL for 24 and 48 hours and the production of TNF-a and expression membrane and soluble TNFRs were investigated. Culture supernatants were collected and levels of TNF-a, IL-10, sTNF-R1 and sTNF-R2 evaluated by ELISA, while levels of nitric oxide (NO) were assessed by the Griess reaction. To determine cell viability the MTT assay was used. Levels of mTNF-R1 and mTNF-R2 were analysed by flow cytometry and the expression of iNOS and Arg-1 were determined by immunocytochemistry. The results show that both antigens induced an increase in TNF-a production (p<0.05) without affecting levels of NO, IL-10, iNOS and Arg-1. Elevated expression of mTNFR1 (p<0,05) in RAW264.7 cells was observed for both antigens after 24h of culture, but only Rv1733 decreased sTNF-R1(p<0,05) in culture supernatants. Expression of mTNF-R2 and detection of sTNF-R2 (p <0.05) was enhanced by Rv1733 after 24h and 48h of culture. These results suggest that the fusion protein ESAT6:CFP10 and the DosR antigen Rv1733 differentially modulate the production of TNF-a and the expression of its receptors on macrophages, which may affect the susceptibility to infection.