Trabajo de Investigación Requisito para optar al Título de Cirujano Dentista / Autor no autoriza el acceso a texto completo de su documento / Entacapone, inhibidor de la catecol-O-metil-transferasa (COMT), representa una nueva generación de fármacos antiparkinson de los cuales hay escasa información de sus efectos en un modelo clásico para el estudio de fármacos antiparkinson, como es el modelo rotacional de ratas con un hemiparkinson inducido por la administración i.c. de la neurotoxina 6-hidroxi-dopamina (6-OH-DA) Metodología: Ratas (n=12) con un hemiparkinson experimental, por lesión unilateral del haz medial cerebral mediante la administración estereotáxica de 8 ug/4 uL de 6-OH- DA, recibieron apomorfina (0,05 mg/kg, s.c.) 2 semanas después de la lesión. Las ratas que tuvieron una conducta rotatoria con apomorfina (n=4) se incluyeron en un protocolo que contempló la administración de anfetamina (2 mg/kg), L-dopa (25 mg/kg), entacapone (20 mg/kg) y, por último, entacapone y L-dopa asociados en las dosis indicadas siempre por vía s.c.. La actividad rotacional se midió mediante un sensor de movimiento y un PC equipado con un software de integración Multicounter ®. Resultados: Anfetamina y entacapone evocaron una rotación ipsilateral a la lesión, mientras que L-dopa lo hizo contralateral a la lesión. No se observó conductas rotacionales por la asociación L-dopa más entacapone. Conclusiones: Entacapone, un inhibidor de la COMT, evoca una actividad rotacional que se explica por la elevación de la concentración de DA a nivel extracelular. La ausencia de actividad rotacional con la asociación entacapone más L-dopa puede explicarse por la anulación de las acciones contrapuestas de cada uno de ellos. Palabras claves: rata, Parkinson, entacapone, conducta rotacional.

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A Doença de Parkinson (DP) é uma doença multifatorial. Seu tratamento é apenas sintomático, o que salienta a importância do estudo de novas terapias. Há evidências de que a excitotoxicidade atribuída ao aumento da atividade glutamatérgica nos núcleos da base seja uma das causas da DP. A guanosina extracelular tem efeitos sobre parâmetros glutamatérgicos e é neuroprotetora: quando administrada oralmente, ela protege ratos contra convulsões induzidas por ácido quinolínico, bem como protege fatias de hipocampo submetidas à combinação de hipóxia e hipoglicemia. O objetivo deste trabalho foi investigar os efeitos do consumo crônico oral de guanosina no modelo animal da DP induzido por 6- hidroxidopamina (6-OHDA). Ratos Wistar machos (250-350g, 100-115 dias de idade) receberam solução de guanosina 0,5mg/ml (n=20) ou água destilada (n=16) para beber durante 4 semanas. Após as duas primeiras semanas de tratamento, receberam infusão de 6-OHDA (5,5 μl, 3 μg/μl) no feixe prosencefálico medial direito. Depois de 2 semanas do término do tratamento, os animais foram submetidos ao Teste de Motricidade sobre Grade (TMG), com duração de 3 minutos e, após uma semana, foram desafiados com metilfenidato (MF) 20 mg/kg, durante 30 minutos, para verificação do grau de lesão dos animais. Imunoistoquímica para TH em células nigrais também foi realizada. Treze de 16 animais do grupo controle apresentaram comportamento rotacional ipsilateral espontâneo no TMG, o que diferiu do grupo tratado, onde apenas nove, dentre os vinte utilizados apresentaram o mesmo comportamento (teste exato de Fisher, p=0,041). Entretanto, não verificamos uma diminuição do número das rotações induzidas por MF 20 mg/kg em animais tratados com guanosina, quando comparado com o grupo controle (teste t para amostras independentes, P = 0,716). Houve tendência a uma menor perda de neurônios positivos para TH na parte compacta da substância nigra no grupo tratado com guanosina (teste t para amostras independentes, p=0,064; grupo controle, n=8, 90%±5, média±E.P.; grupo tratado, n=5, 70%±11). Podemos concluir, a partir desses resultados, que a guanosina previne a expressão comportamental no TMG típica de animais lesionados com 6-OHDA e, talvez, diminua a morte de células nigrais positivas para TH. / Parkinson’s Disease (PD) is a multifactorial disorder and the treatment is only symptomatic, which stresses the importance of studying new therapies. There is some evidences that excitotoxicity attributed to increased glutamatergic activity in the basal ganglia could be one of the causes of PD. Extracellular guanosine has effects on glutamatergic parameters and is neuroprotective: when orally administered, protect against seizures induced by quinolinic acid in rats, and protect brain slices exposed to hypoxia/hypoglycemia. The aim of this work is to investigate the effects of a chronic oral administration of guanosine in the 6-hydroxydopamine (6-OHDA) animal model of PD. Male Wistar rats (250-350 g, 100-115 days old) drank a 0.5 mg/ml guanosine (n=20) or distilled water (n=16) during 4 weeks. After the two first weeks of treatment, they received a stereotaxic infusion of 6-OHDA (16.5 μg, 5.5 μl) into the right medial forebrain bundle (MFB). Two weeks after the end of the treatment, animals were exposed to the footfault apparatus for three minutes; and two weeks after, they were challenged with 20 mg/kg of methylphenidate and were observed for 30 minutes, to verify the degree of lesion. Immunohistochemistry for tyrosine hydroxylase (TH) was also performed. Thirteen of 16 control animals showed spontaneous rotational behavior in the footfault test, which differed from the treated group where only 9 of 20 animals showed the same behavior (Fisher Exact Test, P=0.041). However, the number of rotations induced by MF 20 mg/kg in animals treated with guanosine didn’t change, when compared with the control group (t test for independent samples, P = 0.716). There was a trend towards a smaller decrease of TH-positive nigral neurons in the guanosine-treated animals when compared to controls (independent t test, P=0.064, control group, n=8, 90%+/5. average+/-S.E.M.; treated group, n=5, 70%+/-11) Our results show that guanosine prevents, in some 6-OHDA-lesioned animals, the behavioral pattern of contextinduced rotations in the footfault test. Our preliminary results suggest that guanosine may also have a neuroprotective effect in the 6-OHDA PD model, since we found a trend towards a smaller decrease in the number of TH-positive cells in guanosinetreated animals relative to control animals.

Die Forschung auf dem Gebiet der Parkinson-Erkrankung erlebt einen großen Wandel. Eindeutig ist mittlerweile, dass es zu kurz gefasst wäre diese Erkrankung auf die motorischen Symptome zu beschränken. In den letzten Jahren wurde durch intensive Forschung bewiesen, dass der idiopathische M. Parkinson eine multisystemische Erkrankung ist, welche verschiedene Teile des Nervensystems betreffen kann. Um die zugrundeliegende Pathophysiologie und die Beteiligung des autonomen Nervensystems bei M. Parkinson näher zu untersuchen, wurden für diese Studie 30 Patienten mit idiopathischem M. Parkinson, 19 Patienten mit atypischem Parkinsonsyndrom und 30 gesunde Probanden am Universitätsklinikum Würzburg und an der Paracelsus-Elena-Klinik Kassel rekrutiert. Um Beeinträchtigungen von groß-und kleinkalibrigen Nervenfasern einschätzen zu können, wurden eine Neurografie des N. suralis sowie eine quantitativ sensorische Testung durchgeführt. Zur Bewertung einer möglichen toxischen Komponente von Levodopa gegenüber einer direkten Schädigung peripherer Nerven durch p-α-Synuclein wurden am Vitamin B12 Stoffwechsel beteiligte Proteine im Blut bestimmt. Alle Patienten und Probanden erhielten Hautbiopsien an Unterschenkel, Oberschenkel, Rücken und Finger, um anschließend eine immunhistochemische Aufarbeitung der Präparate durchführen zu können. Einerseits wurde die Beteiligung somatosensibler Nervenfasern mithilfe der Auszählung intraepidermaler Nervenfasern (PGP 9.5) bewertet. Andererseits wurden die Schweißdrüsen auf Pathologien der sympathischen Nervenfasern (VIP, TH, SP, CGRP) und der sudomotorischen Synapsen (SNCA, Synaptophysin, SNAP 25) untersucht. Weiterhin wurde versucht p-α-Synuclein, als Biomarker der Parkinson-Erkrankung, in der Haut nachzuweisen. Positive Ergebnisse konnten hinsichtlich pathologischer Prozesse an den Synapsen erzielt werden. Es zeigte sich sowohl eine Reduktion von nativem α-Synuclein (Unterschenkel, p=0,009 und Rücken, p=0,013), Synaptophysin (Unterschenkel, p=0,007) als auch SNAP 25 (Unterschenkel, p=0,023) an den untersuchten Schweißdrüsen der Patientengruppe. Bei der Untersuchung von SNAP 25 zeigte sich des Weiteren eine negative Korrelation zwischen der SNAP 25 Dichte im Unterschenkel und p-α-Synuclein (p=0,007). Bei der Suche nach p-α-Synuclein wurden beinahe 72% der Parkinson-Patienten positiv getestet, wohingegen keiner der gesunden Probanden p-α-Synuclein in der Haut zeigte. Weiterhin konnte bei 75% der positiv getesteten Patienten mit Multisystematrophie p-α-Synuclein an somatosensiblen Nervenfasern des subepidermalen Plexus nachgewiesen werden, wohingegen es bei den M. Parkinson Patienten nur 13% waren. Die Ergebnisse der zugrundeliegenden Arbeit zeigen, dass die Hautbiopsie als frühdiagnostisches Mittel und in der Differentialdiagnose ein hohes Potenzial hat. Die Erforschung von Pathologien an Synapsen wird in der Zukunft an großer Bedeutung gewinnen und scheint ein wichtiger Ansatz, um die Pathophysiologie des M. Parkinson genauer zu verstehen. Die Hautbiopsie könnte dabei von Vorteil sein, da sich Pathologien in vivo untersuchen lassen und man nicht auf Ergebnisse von Autopsien angewiesen ist. / During the last years it was proved by intensive research that idiopathic parkinson’s disease is multisystemic and can concern different parts of the nervous system. To examine the pathophysiology and the participation of the autonomic nervous system, we recruited 30 patients with idiopathic parkinson’s disease, 19 patients with atypical parkinsonian syndromes and 30 healthy controls from the university medical centre of Würzburg and from the Paracelsus Elena clinic of Kassel for this study. All patients got a neurography of the sural nerve as well as a Quantitative Sensory Testing to estimate involvement of large and small nerve fibres. Proteins, involved in vitamin B12 metabolism, were tested for the assessment of a possible toxic component of Levodopa dosage compared with a direct damage of peripheral nerves by p- α-synuclein. For immunhistochemical analysis all patients and healthy controls received skin biopsies from distal leg, thigh, back and finger. On the one hand the participation of somatosensory nerve fibres was valued with the help of counting up of intraepidermal nerve fibres (PGP 9.5). On the other hand, sweat glands were examined for pathologies of the sympathetic nerve fibres (VIP, TH, SP, CGRP) and the sudomotoric synapses (SNCA, Synaptophysin, SNAP 25). Furthermore we tried to prove that p-α-synuclein could be a biomarker in the skin of patients with idiopathic parkinson’s disease. Positive results could be achieved concerning pathological processes at the synapses. We showed a reduction of native α-synuclein (distal leg, p=0,009 and back, p=0,013), Synaptophysin (distal leg, p=0,007) as well as SNAP 25 (distal leg, p=0,023) in the examined sweat glands of the patient's group. Concerning p-α-synuclein, nearly 72% of patients with parkinson's disease were tested positively, while none of the healthy controls showed deposits. Furthermore we could prove that 75% of the positively tested patients with multiple system atrophy showed p-α-synuclein in somatosensory nerve fibres of subepidermal plexus, while there were only 13% patients with idiopathic parkinson's disease, who showed deposits at this site. The results of this work reveal that skin biopsies have a high potential as early-diagnostic instrument. The investigation of pathologies at synapses will win in great importance and will be necessary to understand the pathophysiology of parkinson's disease. Skin biopsies could be an advantage, because we can examine pathologies in vivo and we don't rely on results of autopsies.

Diss.--Faculty of mathematics and natural sciences--Åbo akademi university, 2001. / Bibliogr. en fin de chap.

Thèse de doctorat : Médecine. Neuroimmunologie : Nantes : 2003. / Thèse : 2003NANT04VS. Bibliogr. f. 101-148, 18-26.

Thèse d'exercice : Pharmacie : Université de Nantes : 2004. / Bibliogr. 4 f.

Thèse d'exercice : Pharmacie : Université de Nantes : 2003. / Bibliogr. f. 113-126 [ réf.].

Reproduction de : Thèse d'exercice : Pharmacie : Nantes : 2009. / Bibliogr.