O presente trabalho contempla o estudo das principais alterações macroscópicas e anatomopatológicas do sistema nervoso central (SNC) encontradas em animais que morreram em decorrência de traumatismo cranioencefálico (TCE). Além da determinação das lesões de golpe, contragolpe e padrão de lesão axonal difusa encontradas nesse tipo de trauma. Inicialmente realizou-se a necropsia dos animais com devido registro fotográfico, descrição das lesões macroscópicas e coleta de material para processamento histológico de rotina e confecção das lâminas coradas em hematoxilina-eosina (HE) para avaliação histopatológica. A imunohistoquímica foi realizada a partir dos mesmos segmentos utilizados nas lâminas de HE, com os anticorpos anti-proteína precursora do β-amiloide (APP), para determinação da lesão axonal, e anti-GFAP e anti-vimentina para avaliação de respostas do tecido nervoso à injúria, como astrocitose e astrogliose. Macroscopicamente as principais lesões observadas caracterizaram-se por lesões focais decorrentes de traumatismo direto, com fraturas de ossos do crânio, laceração de neuroparênquima e contusões. No exame histopatológico as lesões principais caracterizaram-se por hemorragia subdural e em neuroparênquima, congestão, edema tecidual e necrose em variados graus de intensidade e regiões do encéfalo. A lesão axonal foi confirmada pela imunomarcação do βAPP que revelou marcação multifocal de axônios tumefeitos formando esferoides axonais, bulbos axonais ou axônios varicosos em alguns casos. Astrocitose, astrogliose e neovascularização foram observados na maioria dos casos através dos anticorpos GFAP e vimentina. Conclui-se no presente estudo que cães e gatos com TCE apresentaram lesões histologicamente caracterizadas por hemorragias multifocais em meninge e neuroparênquima em diversas regiões, além de edema, necrose e congestão, principalmente. A lesão axonal caracterizou-se pela formação de esferoides axonais, bulbos axonais e longos axônios varicosos. Esta característica foi observada em apenas 2 animais deste estudo, e destes apenas um animal (caso 6) apresentou um padrão de lesão axonal traumática difusa, confirmada pela presença de acentuada marcação pelo anticorpo βAPP. Adicionalmente, foi observada astrocitose e astrogliose em cinco animais, principalmente das áreas próximas às lesões, avaliadas pelos anticorpos GFAP e vimentina. / The present work contemplates the study of the main macroscopic and anatomopathological changes of the central nervous system (CNS) in animals that died due to traumatic brain injury (TBI). Besides the determination of the coup and countercoup and pattern of diffuse axonal injury in this type of trauma. Initially, the animals were necropsied and photographic recorded and macroscopic lesions were described and collect material for histological processing routine and the preparation of slides in hematoxylin-eosin (HE) staining for histopathological evaluation. Immunohistochemistry was performed from the same segments used in the HE slides, with anti-(β-amyloid precursor protein antibodies (βAPP) for determination of axonal lesion, and anti-GFAP and anti-vimentin for evaluation of tissue responses to injury, such as astrocytosis and astrogliosis. Macroscopically, the main lesions observed were focal lesions due to direct trauma, such as skull fractures, neuroparenchyma laceration and contusions. In the histopathological evaluation, the main lesions were characterized by subdural hemorrhage and in neuroparenchyma, congestion, tissue edema and necrosis in varying degrees of intensity and regions of the encephalon. The axonal injury was confirmed by (βAPP immunostaining that revealed multifocal labeled of swellings axons forming axonal spheroids, axonal bulbs or varicose axons in some cases. Astrocytosis, astrogliosis and neovascularization were observed in most cases through the antibodies GFAP and vimentin. In conclusion the present study demonstrate that dogs and cats with TBI presented histologically lesions characterized by multifocal hemorrhage in meninge and neuroparenchyma in different areas, as well as edema, necrosis and congestion, mainly. Axonal injury was determined by axonal swelling, bulbs and varicosities. This characteristic was observed in only 2 animals in this study, and only one of those (case 6) presented pattern of diffuse traumatic axonal injury, confirmed by accentuated (βAPP labeling. Addiotionally, astrocytosis and astrogliosis was observed in five animals, mainly in areas close to lesions, evaluated by GFAP and vimentin antibodies.

Distúrbios do sistema nervoso central (SNC) em ruminantes abrangem um importante grupo de enfermidades responsáveis por grandes perdas econômicas em todo o mundo. As principais doenças neurológicas causadas por bactérias em ruminantes e que envolvem processos inflamatórios incluem listeriose, leptomeningites e meningoencefalites supurativas, abscessos cerebrais e medulares, empiema basilar e neurotuberculose. Esta dissertação teve por objetivo a caracterização epidemiológica, patológica, microbiológica e imuno-histoquímica das doenças inflamatórias de origem bacteriana que afetam o SNC de ruminantes no Rio Grande do Sul. Foi realizado um estudo retrospectivo no período compreendido entre janeiro de 1996 a dezembro de 2015, onde um total de 3.274 bovinos, 596 ovinos e 391 caprinos foram avaliados. Destes, 219 bovinos, 21 ovinos e sete caprinos foram diagnosticados com doenças inflamatórias no SNC. As doenças neurológicas inflamatórias de origem bacteriana totalizaram 60 casos divididos em 34 bovinos, 19 ovinos e sete caprinos, os quais foram subdivididas em: meningoencefalite por L. monocytogenes (oito ovinos, cinco caprinos e quatro bovinos), leptomeningite e meningoencefalite supurativa (14 bovinos, dois caprinos e um ovino), abscessos cerebrais (seis bovinos e dois ovinos) e medulares (sete ovinos), empiema basilar (quatro bovinos e um ovino) e neurotuberculose (seis bovinos). O exame imuno-histoquímica foi realizado em todos os casos diagnosticados com listeriose (anticorpo anti-L. monocytogenes), meningite e meningoencefalite supurativa (anticorpo anti-Escherichia coli) e neurotuberculose (anticorpo anti-Mycobacterium tuberculosis). A meningoencefalite por L. monocytogenes representou a principal enfermidade neurológica em ovinos e caprinos, seguido dos abscessos medulares em ovinos. Nos bovinos, a leptomeningite e meningoencefalite supurativa foi a doença neurológica mais prevalente para a espécie, frequentemente relacionada com a falha na transmissão da imunidade passiva. O empiema basilar, frequentemente diagnosticado em bezerros, está diretamente relacionado com o manejo do desmame interrompido através da utilização de tabuletas nasais. A neurotuberculose causada por Mycobacterium spp. é uma importante doença neurológica em bovinos jovens e deve ser considerada como diagnóstico diferencial de doenças neurológicas. Dos dados analisados neste período, as doenças neurológicas inflamatórias bacterianas representaram um total de 24,3% entre as doenças neurológicas inflamatórias diagnosticadas neste período e, com isso, conclui-se que representam importantes causas de mortalidade para os ruminantes domésticos. / Central nervous system (CNS) diseases are worldwide economically important conditions in ruminants. The main neurological bacterial diseases which involve inflammation in ruminants are listeriosis, suppurative leptomeningitis and meningoencephalitis, brain and spinal cord abscesses, basilar empyema and neurotuberculosis. This study aim to describe the epidemiological, pathological, microbiological and immunohistochemical findings of the bacterial inflammatory diseases that affect the CNS of ruminants in Rio Grande do Sul state. A retrospective study was performed from January 1996 to December 2015, during which samples of 3.274 cattle, 596 sheep and 391 goats were evaluated. Of these, 219 cattle, 21 sheep and seven goats were diagnosed with inflammatory diseases affecting the CNS. The neurological inflammatory bacterial diseases accounted for 60 cases, which corresponded to 34 cases in cattle, 19 in sheep and seven in goats. These were further subdivided in: meningoencephalitis by L. monocytogenes (eigth sheep, five goats and four cattle), suppurative leptomeningitis and meningoencephalitis (14 cattle, two goats and one sheep), brain abscesses (six cattle and two sheep) and spinal cord (seven sheep), basilar empyema (four cattle and one sheep) and neurotuberculosis (six cattle). Immunohistochemical exam was performed in all cases diagnosed as listeriosis (antibody anti-L. monocytogenes), as suppurative leptomeningitis and meningoencephalitis (antibody anti-E. coli), and as neurotuberculosis (antibody anti-Mycobacterium tuberculosis). L. monocytogenes meningoencephalitis was the main neurological disease in sheep and goats, followed by spinal cord abscesses in sheep. In cattle, suppurative leptomeningitis and meningoencephalitis was the most frequent neurological disease, and its ocorrunce is related to the failure in passive immunity transmission. Basilar empyema is frequently diagnosed in calves and is directly related to early weaning handling through the use of nose-flaps. Neurotuberculosis caused by Mycobacterium spp. is an important neurological disease in young cattle and should be consired as a differential diagnosis of granulomatous meningoencephalitis. During the described period, neurological inflammatory bacterial diseases accounted for 24.3% of the neurological inflammatory diseases, and, thus, these are important causes of death in domestic ruminants.

No description available.

No description available.

Distúrbios do sistema nervoso central (SNC) em ruminantes abrangem um importante grupo de enfermidades responsáveis por grandes perdas econômicas em todo o mundo. As principais doenças neurológicas causadas por bactérias em ruminantes e que envolvem processos inflamatórios incluem listeriose, leptomeningites e meningoencefalites supurativas, abscessos cerebrais e medulares, empiema basilar e neurotuberculose. Esta dissertação teve por objetivo a caracterização epidemiológica, patológica, microbiológica e imuno-histoquímica das doenças inflamatórias de origem bacteriana que afetam o SNC de ruminantes no Rio Grande do Sul. Foi realizado um estudo retrospectivo no período compreendido entre janeiro de 1996 a dezembro de 2015, onde um total de 3.274 bovinos, 596 ovinos e 391 caprinos foram avaliados. Destes, 219 bovinos, 21 ovinos e sete caprinos foram diagnosticados com doenças inflamatórias no SNC. As doenças neurológicas inflamatórias de origem bacteriana totalizaram 60 casos divididos em 34 bovinos, 19 ovinos e sete caprinos, os quais foram subdivididas em: meningoencefalite por L. monocytogenes (oito ovinos, cinco caprinos e quatro bovinos), leptomeningite e meningoencefalite supurativa (14 bovinos, dois caprinos e um ovino), abscessos cerebrais (seis bovinos e dois ovinos) e medulares (sete ovinos), empiema basilar (quatro bovinos e um ovino) e neurotuberculose (seis bovinos). O exame imuno-histoquímica foi realizado em todos os casos diagnosticados com listeriose (anticorpo anti-L. monocytogenes), meningite e meningoencefalite supurativa (anticorpo anti-Escherichia coli) e neurotuberculose (anticorpo anti-Mycobacterium tuberculosis). A meningoencefalite por L. monocytogenes representou a principal enfermidade neurológica em ovinos e caprinos, seguido dos abscessos medulares em ovinos. Nos bovinos, a leptomeningite e meningoencefalite supurativa foi a doença neurológica mais prevalente para a espécie, frequentemente relacionada com a falha na transmissão da imunidade passiva. O empiema basilar, frequentemente diagnosticado em bezerros, está diretamente relacionado com o manejo do desmame interrompido através da utilização de tabuletas nasais. A neurotuberculose causada por Mycobacterium spp. é uma importante doença neurológica em bovinos jovens e deve ser considerada como diagnóstico diferencial de doenças neurológicas. Dos dados analisados neste período, as doenças neurológicas inflamatórias bacterianas representaram um total de 24,3% entre as doenças neurológicas inflamatórias diagnosticadas neste período e, com isso, conclui-se que representam importantes causas de mortalidade para os ruminantes domésticos. / Central nervous system (CNS) diseases are worldwide economically important conditions in ruminants. The main neurological bacterial diseases which involve inflammation in ruminants are listeriosis, suppurative leptomeningitis and meningoencephalitis, brain and spinal cord abscesses, basilar empyema and neurotuberculosis. This study aim to describe the epidemiological, pathological, microbiological and immunohistochemical findings of the bacterial inflammatory diseases that affect the CNS of ruminants in Rio Grande do Sul state. A retrospective study was performed from January 1996 to December 2015, during which samples of 3.274 cattle, 596 sheep and 391 goats were evaluated. Of these, 219 cattle, 21 sheep and seven goats were diagnosed with inflammatory diseases affecting the CNS. The neurological inflammatory bacterial diseases accounted for 60 cases, which corresponded to 34 cases in cattle, 19 in sheep and seven in goats. These were further subdivided in: meningoencephalitis by L. monocytogenes (eigth sheep, five goats and four cattle), suppurative leptomeningitis and meningoencephalitis (14 cattle, two goats and one sheep), brain abscesses (six cattle and two sheep) and spinal cord (seven sheep), basilar empyema (four cattle and one sheep) and neurotuberculosis (six cattle). Immunohistochemical exam was performed in all cases diagnosed as listeriosis (antibody anti-L. monocytogenes), as suppurative leptomeningitis and meningoencephalitis (antibody anti-E. coli), and as neurotuberculosis (antibody anti-Mycobacterium tuberculosis). L. monocytogenes meningoencephalitis was the main neurological disease in sheep and goats, followed by spinal cord abscesses in sheep. In cattle, suppurative leptomeningitis and meningoencephalitis was the most frequent neurological disease, and its ocorrunce is related to the failure in passive immunity transmission. Basilar empyema is frequently diagnosed in calves and is directly related to early weaning handling through the use of nose-flaps. Neurotuberculosis caused by Mycobacterium spp. is an important neurological disease in young cattle and should be consired as a differential diagnosis of granulomatous meningoencephalitis. During the described period, neurological inflammatory bacterial diseases accounted for 24.3% of the neurological inflammatory diseases, and, thus, these are important causes of death in domestic ruminants.

Distúrbios do sistema nervoso central (SNC) em ruminantes abrangem um importante grupo de enfermidades responsáveis por grandes perdas econômicas em todo o mundo. As principais doenças neurológicas causadas por bactérias em ruminantes e que envolvem processos inflamatórios incluem listeriose, leptomeningites e meningoencefalites supurativas, abscessos cerebrais e medulares, empiema basilar e neurotuberculose. Esta dissertação teve por objetivo a caracterização epidemiológica, patológica, microbiológica e imuno-histoquímica das doenças inflamatórias de origem bacteriana que afetam o SNC de ruminantes no Rio Grande do Sul. Foi realizado um estudo retrospectivo no período compreendido entre janeiro de 1996 a dezembro de 2015, onde um total de 3.274 bovinos, 596 ovinos e 391 caprinos foram avaliados. Destes, 219 bovinos, 21 ovinos e sete caprinos foram diagnosticados com doenças inflamatórias no SNC. As doenças neurológicas inflamatórias de origem bacteriana totalizaram 60 casos divididos em 34 bovinos, 19 ovinos e sete caprinos, os quais foram subdivididas em: meningoencefalite por L. monocytogenes (oito ovinos, cinco caprinos e quatro bovinos), leptomeningite e meningoencefalite supurativa (14 bovinos, dois caprinos e um ovino), abscessos cerebrais (seis bovinos e dois ovinos) e medulares (sete ovinos), empiema basilar (quatro bovinos e um ovino) e neurotuberculose (seis bovinos). O exame imuno-histoquímica foi realizado em todos os casos diagnosticados com listeriose (anticorpo anti-L. monocytogenes), meningite e meningoencefalite supurativa (anticorpo anti-Escherichia coli) e neurotuberculose (anticorpo anti-Mycobacterium tuberculosis). A meningoencefalite por L. monocytogenes representou a principal enfermidade neurológica em ovinos e caprinos, seguido dos abscessos medulares em ovinos. Nos bovinos, a leptomeningite e meningoencefalite supurativa foi a doença neurológica mais prevalente para a espécie, frequentemente relacionada com a falha na transmissão da imunidade passiva. O empiema basilar, frequentemente diagnosticado em bezerros, está diretamente relacionado com o manejo do desmame interrompido através da utilização de tabuletas nasais. A neurotuberculose causada por Mycobacterium spp. é uma importante doença neurológica em bovinos jovens e deve ser considerada como diagnóstico diferencial de doenças neurológicas. Dos dados analisados neste período, as doenças neurológicas inflamatórias bacterianas representaram um total de 24,3% entre as doenças neurológicas inflamatórias diagnosticadas neste período e, com isso, conclui-se que representam importantes causas de mortalidade para os ruminantes domésticos. / Central nervous system (CNS) diseases are worldwide economically important conditions in ruminants. The main neurological bacterial diseases which involve inflammation in ruminants are listeriosis, suppurative leptomeningitis and meningoencephalitis, brain and spinal cord abscesses, basilar empyema and neurotuberculosis. This study aim to describe the epidemiological, pathological, microbiological and immunohistochemical findings of the bacterial inflammatory diseases that affect the CNS of ruminants in Rio Grande do Sul state. A retrospective study was performed from January 1996 to December 2015, during which samples of 3.274 cattle, 596 sheep and 391 goats were evaluated. Of these, 219 cattle, 21 sheep and seven goats were diagnosed with inflammatory diseases affecting the CNS. The neurological inflammatory bacterial diseases accounted for 60 cases, which corresponded to 34 cases in cattle, 19 in sheep and seven in goats. These were further subdivided in: meningoencephalitis by L. monocytogenes (eigth sheep, five goats and four cattle), suppurative leptomeningitis and meningoencephalitis (14 cattle, two goats and one sheep), brain abscesses (six cattle and two sheep) and spinal cord (seven sheep), basilar empyema (four cattle and one sheep) and neurotuberculosis (six cattle). Immunohistochemical exam was performed in all cases diagnosed as listeriosis (antibody anti-L. monocytogenes), as suppurative leptomeningitis and meningoencephalitis (antibody anti-E. coli), and as neurotuberculosis (antibody anti-Mycobacterium tuberculosis). L. monocytogenes meningoencephalitis was the main neurological disease in sheep and goats, followed by spinal cord abscesses in sheep. In cattle, suppurative leptomeningitis and meningoencephalitis was the most frequent neurological disease, and its ocorrunce is related to the failure in passive immunity transmission. Basilar empyema is frequently diagnosed in calves and is directly related to early weaning handling through the use of nose-flaps. Neurotuberculosis caused by Mycobacterium spp. is an important neurological disease in young cattle and should be consired as a differential diagnosis of granulomatous meningoencephalitis. During the described period, neurological inflammatory bacterial diseases accounted for 24.3% of the neurological inflammatory diseases, and, thus, these are important causes of death in domestic ruminants.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Canine distemper is one of the most prevalent viral diseases of dogs. Several cases are diagnosed in a daily basis in private practices and veterinary hospitals around the country. Most cases of neurological manifestation of canine distemper are fatal and the diagnosis confirmation by histopathology is often necessary. Canine distemper encephalitis is the main cause of death or reason for euthanasia in dogs necropsied at the Laboratório de Patologia Veterinária (LPV) of the Universidade Federal de Santa Maria (UFSM). Consequently the large number of canine distemper cases in the LPV-UFSM files prompted the performance of a retrospective study consisting of 620 neurologic cases of canine distemper which served as the basis for a prospective study on the neuro-histopathologic aspects of the disease. The current study was carried out to perform a detailed histopathologic investigation on the changes in the central nervous system (CNS) of dogs affect by canine distemper aiming to help students of veterinary pathology and veterinary pathologists in the correct diagnose of this disease. Seventy dogs necropsied at the LPV-UFSM and with diagnosis of canine distemper confirmed by the finding of characteristic inclusion bodies in the CNS were included in the study. In order to determine the prevalence of the lesions, several anatomical regions were selected from the brain and spinal cord; these sites were consistently microscopically examined in each case. Overall, most affected anatomical regions were, in decreasing order of frequency: cerebellum (91.4%), diencephalon (78.6%), frontal lobe (75.7%), pons (72.9%) and mesencephalon (70.0%). Demyelination was the most prevalent lesion; it was observed in 91.4% of the cases and was located mainly in the cerebellum (88.6%), pons (65.7%) and diencephalon (61.4%). The five structures most affected by demyelination were: roof of the fourth ventricle (68.6%), cerebellar folia (61.4%), cerebellar medulla (61.4%), cervical spinal cord (46.3%) and mesencephalic tegment (40.0%). Other lesions and their prevalence were non-suppurative encephalitis (70.0%), non-suppurative leptomeningitis (44.3%), non-suppurative myelitis (35.2%), encephalomalacia (31.4%), nonsuppurative myelitis (18.5%), laminar cortical necrosis (17.1%), myelomalacia (13.0%), nonsuppurative ependymitis (7.1%) and plexochoroiditis (1.4%). The occurrence of the inclusion bodies in the cells of the CNS was: astrocytes (82.8%), neurons (44.3%), gemistocytes (31.4%), ependymal cells (22.8%), meningothelial cells (4.3%) and cells of the choroids plexus (1.4%). / A cinomose é uma das doenças virais mais prevalentes em cães. Muitos cães são diagnosticados com a doença diariamente em clínicas e hospitais veterinários de todo o país. A forma neurológica da cinomose é fatal na grande maioria das vezes, e a confirmação do diagnóstico clínico através do exame histopatológico se faz necessária em muitos casos. A encefalite por cinomose representa a principal causa de morte ou razão para eutanásia de cães necropsiados no Laboratório de Patologia Veterinária (LPV) da Universidade Federal de Santa Maria (UFSM). Com isso, o grande número de casos de cinomose arquivados no LPVUFSM proporcionou a realização de um estudo retrospectivo de 620 casos da forma neurológica da doença que serviu como base para a realização de um estudo neurohistopatológico prospectivo. O presente estudo tem como objetivo realizar uma investigação histopatológica detalhada das alterações no sistema nervoso central (SNC) de cães com cinomose para auxiliar estudantes de patologia e patologistas no diagnóstico da doença. Para isso, foram selecionados 70 casos da forma neurológica da cinomose confirmada pelo achado dos corpúsculos de inclusão característicos no SNC em cães necropsiados no LPV-UFSM. Para determinar a prevalência das lesões, diversas regiões anatômicas do encéfalo e medula espinhal foram selecionadas e sistematicamente examinadas histologicamente em cada caso. As regiões anatômicas mais afetadas, em ordem decrescente, independentemente do tipo de lesão, foram: cerebelo (91,4%), diencéfalo (78,6%), lobo frontal (75,7%), ponte (72,9%) e mesencéfalo (70,0%). Desmielinização foi a lesão mais prevalente, observada em 91,4% dos casos, localizada principalmente no cerebelo (88,6%), ponte (65,7%) e diencéfalo (61,4%). As cinco estruturas anatômicas mais afetadas pela desmielinização foram: teto do quarto ventrículo (68,6%), folhas cerebelares (61,4%), substância medular do cerebelo (61,4%), medula espinhal cervical (46,3%) e tegmento mesencefálico (40,0%). A prevalência das outras lesões foi: encefalite não-supurativa (70,0%), leptomeningite não-supurativa (44,3%), mielite não-supurativa (35,2%), encefalomalacia (31,4%), meningomielite não-supurativa (18,5%), necrose laminar cortical (17,1%), mielomalacia (13,0%), ependimite não-supurativa (7,1%) e plexocoroidite não-supurativa (1,4%). A ocorrência dos corpúsculos de inclusão nas células do SNC foi: astrócitos (82,8%), neurônios (44,3%), gemistócitos (31,4%), células do epêndima (22,8%), células meningoteliais (4,3%) e células do plexo coróide (1,4%).

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Light and ultraestructural changes of central and peripheral nervous system lesions in mice KO for connexin-32 and submitted to the ethidium bromide gliotoxic demyelinating model are described. Their KO condition was tested with PCR and a negative connexin-32 labelling was performed by immunofluorescence. The experimental animals were C57BL/6 normal mice and C57BL/6 KO for connexin-32. For all groups the animals were maintained in cages of 5 individuals within a temperature controlled room and had ration and filtered water ad libitum. A single local injection of either 0,1% ethidium bromide in normal saline (5 μl in the brainstem and 1 μl in the sciatic nerve) or normal saline was performed as described for Wistar rats. The injected mice were observed daily until euthanasia was performed at 24, 48 hours and 3, 7, 15, 21 and 30 days after injection. The mice were perfused through the heart with either neutral 10% formalin or 2,5% glutaraldehyde. Histochemical, immunohistochemical, immunofluorescence and transmission electron microscopic methods were used to analyze the development of the lesions after differentiated processing. Hematoxylin- eosine, luxol fast blue and toluidine blue methods and immunolabelling with anti-GFAP, anti-CNPase, anti-S100 protein and anti-OSP, anti Cx32 and anti Cx43 antibodies were used. Within the CNS the lesions showed an acute degenerative phase with disappearance of glial cells, and myelin sheaths were withdrawn by a scant number of macrophages. In KO mice some granulocytes were detected within the lesions in tight contact with decaying myelin sheaths. Remyelination was carried out by oligodendrocytes since no Schwann cells were seen during the regenerating process of KO mice. Occasional remyelinating Scwann cells were seen in normal mice. For the sciatic nerves, Schwann cells initially showed signs of intoxication and rejected their sheaths; after seven days, some thin newly formed myelin sheaths with uneven compaction and redundant loops (tomacula) were conspicuous. Mast cells degranulated or not were seen in all BE- induced lesions and after saline injection. It is concluded that the repair of the CNS demyelinated lesions differs from the observed in normal and immunosupressed rats because Schwann cells remyelination was absent; the absence of connexin-32 may have caused that absence. The regeneration of lost myelin sheaths within the PNS followed the pattern already reported for this model in other species. It is suggested that the absence of connexin-32 determined the different repair of the myelin sheaths within the CNS whereas for the PNS, the normal pattern of tissue response might be due to the early age of the injected mice. / São descritas as alterações de microscopia de luz e ultra-estruturais induzidas pelo brometo de etídio no sistema nervoso central e periférico de camundongos KO para conexina 32. O genótipo KO foi testado por PCR e confirmado por imunofluorescência negativa para conexina 32. Os animais dos experimentos foram camundongos C57BL-6 normais (controles) e KO para conexina 32. Todos os camundongos foram mantidos em gaiolas de 5 indivíduos em sala climatizada e receberam ração e água à vontade. Uma única injeção de BE 0,1% em salina 0,9% ou de salina 0,9% (5 μl na cisterna basal e 1μl no nervo ciático) foi realizada como descrita em ratos Wistar. Os camundongos eram observados diariamente até ser realizada a eutanásia às 24 e 48 h, 3, 7, 15, 21 e 30 dias após a injeção. Os camundongos foram perfundidos através do coração; um grupo com glutaraldeído 2,5% visando o processamento para microscopia eletrônica; um outro grupo com solução salina com EDTA e posterior fixação em metacarn para inclusão em parafina. As amostras incluídas em parafina foram analisadas através dos métodos de hematoxilina e eosina, luxol fast blue e azul de toluidina. Foram realizadas imunoistoquímica e imunofluorescência visando a marcação de GFAP, CNPase, OSP, S100, e Cx43 e Cx32, respectivamente. As lesões do SNC eram discretas e tiveram uma fase ativa com desaparecimento das células gliais; os debris celulares e de mielina foram retirados por um reduzido número de fagócitos. Nos camundongos KO foram vistos granulócitos em estreito contato com bainhas de mielina em degradação. A remielinização dos axônios desmielinizados foi realizada exclusivamente por oligodendrócitos nos camundongos KO; nos camundongos normais, ocasionais células de Schwann podiam ser encontradas remielinizando axônios do SNC. No nervo ciático, as células de Schwann intoxicadas rejeitaram seus internodos de mielina; após sete dias, finas bainhas reparadas eram encontradas, com compactação irregular da mielina e alças redundantes (tomacula). Mastócitos, desgranulados ou não, eram vistos nas lesões do BE e após a injeção de solução salina. Conclui-se que o reparo das lesões do SNC difere do observado em ratos normais e imunossuprimidos devido à ausência de remielinização por células de Schwann; a falta de expressão da Cx 32 e o tamanho reduzido das lesões podem ter contribuído para essa ausência. A regeneração das bainhas perdidas no SNP obedeceu ao padrão descrito para esse modelo em outras espécies. Sugere-se que a ausência da Cx 32 não afetou o reparo do SNP devido à idade precoce dos animais.

Amyotrophe Lateralsklerose (ALS) ist eine neurodegenerative Erkrankung, bei welcher speziell erste (kortikospinal) und zweite (spinal) Motorneurone (MN) von Neurodegeneration betroffen sind. Gegenwärtig bleibt ALS eine unheilbare Erkrankung. Der Tod tritt durchschnittlich 2 bis 5 Jahre nach Auftreten der Symptome ein. Circa 90% der Fälle treten sporadisch auf (sALS), während 10% familiär sind (fALS). Es ist von großem Interesse monogenetische Formen der fALS zu untersuchen um zugrundeliegende Pathologien und Mechanismen zu verstehen. Bislang wurden über 20 Gene mit ALS in Verbindung gebracht, einschließlich Fused in sarcoma (FUS) und Chromsosome 9 open reading frame (C9ORF72). Circa 4% der fALS Fälle sind durch dominante Mutationen in FUS verursacht und repräsentieren damit die dritthäufigste Form der fALS in Deutschland. Die G4C2 hexanucleotide repeat expansion (HRE) in C9ORF72 ist die häufigste Ursache für ALS und Frontotemporale Demenz (FTD). ALS Patienten unterscheiden sich erheblich in der Präsentation ihrer klinischen Symptome wie Ausbruchsort, Progressionsrate und Auftreten kognitiver Störungen. Diese Faktoren sind auch stark abhängig von der zugrundeliegenden Mutation in fALS. Ziel dieser Doktorarbeit ist die Modellierung von FUS- und C9ORF72-assozierter ALS in einem krankheits-relevanten in vitro Model von speziell kortikaler Neuropathologie mit Hilfe von Patienten-spezifischen iPSZs. Die Hypothese der vorliegenden Arbeit ist das in einer Zelltyp-abhängigen Art und Weise zugrundeliegende Erkrankungsmechanismen in kortikalen vs. spinalen Neuronen unterschiedlich betroffen sind. Humane iPSZ, generiert von gesunden Kontrollen und ALS Patienten mit FUS oder C9ORF72 Mutation, wurden für die gerichtete kortikale und spinale Differenzierung genutzt. Zusätzlich wurden zwei neue FUS-WT- und FUS-P525L-EGFP-markierte isogene Linien mittels CRISPR/Cas9n Technik generiert. Methoden basierend auf Immunfluoreszenz Färbungen und Lebendzell-Mikroskopie wurden angewendet um Krankheits-relevante Proteine, DNA Schäden und axonale Organell-Mobilität zu analysieren. In diesem Projekt konnte ein deutlicher Zelltyp-abhängiger Effekt auf analysierte Phänotypen beobachtet werden, während ALS-assoziierte Mutationen scheinbar nur geringfügige Effekte zeigten. Dementsprechend wurde ein Zelltyp-abhängiger Anstieg des basalen DNA Schadens in kortikalen Astrozyten vs. Neuronen und spinalen vs. kortikalen Neuronen detektiert. Jedoch konnte in FUS oder C9ORF72 mutierten kortikalen Zellen kein erhöhter DNA Schaden nachgewiesen werden, wie es zuvor in spinalen MN beobachtet wurde. Des Weiteren beeinflussen FUS Mutationen die Rekrutierung von FUS zu DNA-geschädigten Stellen, die Organell-Mobilität und die zytoplasmatische Fehllokalisation des Proteins in Abhängigkeit vom Zelltyp. In kortikalen Neuronen wurde in Bezug auf die Rekrutierung von mutiertem FUS und Organell-Mobilität nur leichte Mutations-abhängige und wesentlich schwächer ausgeprägte Effekte beobachtet als in spinalen MN. Zusammenfassend kann gesagt werden, dass Patienten-spezifische Zellmodelle ein wichtiges Instrument in der ALS Forschung sind und das vor allem Unterschiede zwischen kortikalen und spinalen MN weiter untersucht werden müssen, um zugrundeliegende Krankheits-relevante Mechanismen zu entschlüsseln und wie diese zum Fortschreiten der Erkrankung beitragen / Amyotrophic lateral sclerosis (ALS) is a of neurodegenerative diseases, in which neurodegeneration specifically affects upper (corticospinal) and lower (spinal) motor neurons (MNs). At present, ALS remains an incurable disease. Death occurs on average 2 to 5 years after symptom onset. About 90% are sporadic cases (sALS) and 10% are familial cases (fALS). It is of great interest to investigate monogenetic forms causing fALS to understand its underlying disease pathologies and mechanisms. Over 20 genes have been linked to ALS until now, including Fused in sarcoma (FUS) and Chromosome 9 open reading frame 72 (C9ORF72). About 4% of fALS cases are caused by dominant mutations within FUS, representing the third most common fALS form in Germany. The G4C2 hexanucleotide repeat expansion (HRE) in the C9ORF72 gene is the most common cause for ALS and Frontotemporal dementia (FTD). ALS patients differ significantly in their presentation of clinical symptoms, including site of onset, rate of progression, and presence of cognitive dysfunction. Those factors were also shown to highly depend on the underlying mutation in fALS cases. Aim of this thesis work is the modeling of FUS- and C9ORF72-associated ALS in a disease-related in vitro model of particularly cortical neuropathology using patient-derived iPSCs. The hypothesis of the current work is that underlying disease mechanisms do differentially affect cortical vs. spinal neurons and act in a cell type-dependent manner. Human iPSCs derived from healthy controls and ALS patients carrying mutations within FUS or C9ORF72 were used for directed cortical and spinal differentiation. Additionally, two new FUS-WT- and FUS-P525L-EGFP-tagged isogenic iPSC lines were generated by CRISPR/Cas9n gene editing. Immunofluorescence staining and live cell imaging approaches were implemented to analyze disease-associated proteins, DNA damage, and axonal trafficking. Within this project, a clear cell type-dependent effect on analyzed phenotypes was observed, while ALS-associated mutations seemed to have only minor effects. Accordingly, cell type-dependent increased basal DNA damage levels in cortical astrocytes vs. neurons and spinal vs. cortical neurons were detected. However, FUS or C9ORF72 mutant cortical cells do not recapitulate increased DNA damage levels as they have been observed in spinal MNs. Furthermore, FUS mutation affected recruitment to DNA damage sites, axonal trafficking, and cytoplasmic mislocalization differentially, depending on the analyzed cell type. In cortical neurons, recruitment and trafficking of mutant FUS showed only slight mutation-dependent effects and also less pronounced phenotypes than observed in spinal MNs. In conclusion, patient-specific cellular models are an important tool in ALS research and particularly differences between cortical and spinal MNs need to be further investigated to decipher underlying disease mechanisms, the interplay of cell types affected by the disease, and how they participate in disease progression.

Clinical variants of dementia are limiting their diagnosis and can leads to underdiagnosing or substitution of two different diseases with the same symptomatology. The aim of this study is a better understanding of a factors involved in the clinical variability of rare dementias. Progressive supranuclear palsy and Gerstmann-Sträussler-Scheinker syndrome caused by mismatch mutation P102L in Prion protein are used as model diseases. In this thesis, we firstly demonstrate the influence of the distribution of neuropathology and its spread on the clinical phenotype of the disease. Although a single neurodegenerative disease increases the risk of neurodegenerative comorbidity, this other neuropathology does not affect the phenotypic presentation of the primary disease. Monogenetically inherited proteinopathies can have a different clinical subtype, which is not only conditioned by causal protein polymorphisms, but can be influenced by the wild type allele of causal protein. A more accurate understanding of the symptomatic variability in dementias will allow a better focus of a drug studies and, in the future a treatment, but it will also lead to a better understanding of the pathogenesis of neurodegenerative diseases. Keywords: dementia, Progressive supranuclear palsy, Gerstmann-Sträussler-Scheinker...