Die Osteoarthrose (OA) des Ellbogengelenks ist eine der wichtigsten Gelenkerkrankungen des Hundes, die sich aufgrund erblich bedingter Fehlstellung der Gelenke sekundär häufig schon bei sehr jungen Hunden entwickelt und zu einer verminderten Lebensqualität der Tiere führt. Im Laufe der Erkrankung kommt es zu einer Schädigung des Gelenkknorpels, der an Elastizität und Spannkraft verliert und seine Funktion, nicht mehr ausüben kann. Eine Ablösung von Knorpelfragmenten und Entzündungen im Gelenk können die Zerstörung des Gelenkknorpels beschleunigen. Die OA ist nicht heilbar. Das Therapieziel ist eine möglichst vollständige Schmerzreduktion und die Verbesserung der Lebensqualität. Eine chirurgische Denervation ist im Ellbogengelenk des Hundes ohne Traumatisierung anatomischer Strukturen nicht möglich. Ziel der vorliegenden Arbeit war es, erstmalig einen experimentellen Ansatz zur chemischen Denervation sensibler Gelenkafferenzen im Ellbogengelenk beim Beagle mit dem Neurotoxin OX7-Saporin durchzuführen. Zusätzlich wurden Substanzen aus der Humanmedizin zur Schmerzreduktion und symptomatischen Therapie der OA am Hund erprobt und der Therapieerfolg evaluiert. Im Rahmen eines Therapieversuches in der Klinik für Kleintiere der Universität Leipzig wurde bei zwei, an OA erkrankten Hunden Capsaicin einmalig intraartikulär injiziert. Der erste Hund erhielt intraartikulär 250 mg Capsaicin. Dieser Patient zeigte Nebenwirkungen in Form einer Herz- und Atemfrequenzerhöhung, starker Schmerzhaftigkeit und neurologischen Symptomen. Der zweite Hund wurde mit 83 mg Capsaicin behandelt und zeigte keine Nebenwirkungen. Sowohl die Bewertung der Besitzer als auch die orthopädische Untersuchung ergaben keine Verbesserung der Lahmheit. Vier kranken Hunden wurde ebenfalls im Rahmen eines Therapieversuches Botox (Botulinumtoxin A) intraartikulär einmalig injiziert. Drei Hunde wurden mit 50 Einheiten, ein Hund mit 100 Einheiten Botox behandelt. Die Applikation von Capsaicin und Botox führten zu keiner Verbesserung der Symptomatik. In der Humanmedizin ist die Schmerzbehandlung mit Capsaicin und Botox erfolgreich. Deshalb sollten diese Therapieansätze auch für die Anwendung beim Hund weiter optimiert werden. Das Neurotoxin OX7-Saporin wurde in einer Dosierung von 100 µg erstmalig in das Ellbogengelenk von drei Beagle-Hündinnen appliziert, um eine sensible Denervation des Gelenkes zu erreichen. Der retrograde Transport des Ribosomen-inaktivierenden Proteins in die Perikarya der Spinalganglienzellen, die das Ellbogengelenk sensibel innervieren, sollte eine selektive Zerstörung der Neurone durch das Neurotoxin bewirken. Der retrograde Tracer Fluoro-Gold wurde, zur Kontrolle der Wirkung des Neurotoxins, nach einer Wartezeit von 15 Tagen intraartikulär in das linke und das rechte Ellbogengelenk appliziert. Fluoro-Gold kann ausschließlich von intakten Nervenfasern transportiert werden. Die Ganglien C4 - Th3 wurden bilateral zur histologischen Auswertung entnommen und in Paraffin eingebettet. Zur Bestimmung der Gesamtneuronenanzahl wurden alle Neurone in jedem dritten Schnitt eines Ganglions gezählt. Die Summe dieser Nervenzellen ergab die Gesamtneuronenanzahl eines Ganglions. Die Einlagerung von Lipofuszin führte zu einer starken Autofluoreszenz im Zytoplasma der Neuronen. Alle Schnitte wurden mit Sudan-Schwarz gefärbt, um die Autofluoreszenz von der retrograden FG-Markierung zu unterscheiden. Die FG-Markierung konnte nur in den Ganglien C6 und Th1 im Zytoplasma sehr weniger Neuronen dokumentiert werden. Der histologische Nachweis einer Neurodegeneration nach Applikation des Neurotoxins OX7-Saporin in das linke Ellbogengelenk war negativ. In den untersuchten Spinalganglien wurden intakte Nervenzellen nachgewiesen. Auch die bilaterale intraartikuläre Injektion des retrograden Tracers FG war nicht erfolgreich. Aus den Ergebnissen dieser experimentellen Ansätze wird zusammenfassend geschlussfolgert: Ø Vor der Durchführung zukünftiger experimenteller Untersuchungen beim Hund muss die optimale Konzentration und die Wartezeit für das Neurotoxins OX7-Saporin ermittelt werden. Ø Die Rezeptoren für OX7-Saporin müssen an den Nervenzellen des Hundes zweifelsfrei nachgewiesen werden, bevor das Neurotoxin im caninen Tiermodell eingesetzt werden kann. Ø Die optimale Konzentration des Fluoreszenzfarbstoffes FG für intraartikuläre Injektionen und dessen retrograde Transportgeschwindigkeit beim Hund müssen ermittelt werden, bevor dieser Tracer wieder im Tierexperiment eingesetzt werden kann. Ø FG wurde als 1,8%ige Lösung in das Ellbogengelenk beim Beagle appliziert. Diese Konzentration war wahrscheinlich zu gering, um eine retrograde Markierung der sensiblen Neurone in den entsprechenden Spinalganglienzellen nachzuweisen. Ø Die intraartikuläre Applikation des Neurotoxins OX7-Saporin führte nicht zur selektiven Neurodegeneration. Eine Aussage zur Schmerzausschaltung und symptomatischen Therapie der OA des Hundes mit OX7-Saporin ist deshalb nicht möglich.

This dissertation will examine the phenotype of pain facilitatory neurons in the rostral ventromedial medulla (RVM) and its role in neuropathic pain states. Activation of the descending facilitation pathways might be the result of plasticity in the RVM that is driven, at least in part, by the presence and activity of cholecystokinin type-2 receptors (CCK2R) mRNA expressing neurons. The expression of either opioid mu receptors (MOR) or CCK2R mRNA in the RVM was confirmed by in situ hybridization (ISH). Pretreatment with CCK8(s)-saporin resulted in a significant loss of CCK2R mRNA positive cells in the RVM, concomitant with a blockade of CCK8(s) induced hyperalgesia. The same treatment also significantly reduced the number of neurons labeled for MOR mRNA, hinting that MOR and CCK2R mRNA signals may be co-localized in some RVM cells. Consistent with these data, pretreatment with dermorphin-saporin significantly reduced the number of MOR mRNA labeled cells in the RVM, blocked RVM CCK8(s) induced hyperalgesia and reduced the number of CCK2R mRNA positive cells in the RVM. The co-localization was further confirmed by a dual label ISH approach using 35S-labeled CCK2R and Digoxigenin-labeled MOR riboprobes. Data showed that over 80% of labeled RVM neurons co-expressed both MOR and CCK2R mRNA, ~15% expressed only CCK2R mRNA, and very few cells expressed only MOR mRNA. The above findings may suggest that elimination of CCK2R mRNA expressing neurons result in removal of the driving force for descending facilitation from RVM, hereby block the development of neuropathic pain. Rats pretreated with CCK8(s)-saporin conjugates had a full reversal of thermal sensory threshold and partial reversal of tactile threshold starting at day 5 after SNL. The lesion effects of RVM CCK-SAP were evaluated by ISH. Comparing to saporin pretreated groups, CCK8(s)-saporin pretreatment significantly reduced the numbers of CCK2R mRNA labeled neurons within RVM. The data suggest that selective ablation of CCK2R mRNA expressing cells in RVM is sufficient to block the development of neuropathic pain, and thus confirm the hypothesis that CCK2R mRNA expressing cells may be an important player in descending facilitation from RVM as a mechanism of neuropathic pain.

Les thérapies ciblées utilisent des agents thérapeutiques qui interfèrent specifiquement avec les molécules nécessaires pour la croissance et la progression tumorale. Les chimiothérapies classiques sont toxiques pour les cellules qui se divisent rapidement du à leur interaction avec les cellules en division. Le premier but des thérapies ciblées est de combattre plus précisement les cellules cancereuses et ainsi éviter les effets indesirables.La thérapie anti-cancereuse utilisant les anticorps a été développé depuis environ 15 ans et est actuellement une des plus efficaces des thérapies ciblées. Dans certains cas des anticorps monoclonaux sont conjugués avec des isotopes radioactifs ou des toxines pour permettre une délivrance ciblée de ces derniers dans les cellules cancereuses. De plus en plus, pour remplacer les anticorps, les thérapies ciblées utilisent des peptides ou des acides nucleiques comme agent ciblant.Dans ce travail, nous avons utilisé des stratégies diverses de ciblage pour permettre l’internalisation de substance toxique (une toxine ou son gène) specifiquement dans les cellules cancereuses. Nous avons travaillé sur le modèle du glioblastome.Notre groupe a publié un article décrivant l’utilisation du domaine PDZ d’une proteine hCASK permettant la liaison a un biomarqueur surexprimé dans les cellules cancereuses. En effet, ce domaine PDZ est capable de se lier à la partie C-terminale de la proteine CD98. Le produit de fusion hCASK-PDZ a été génétiquement lié a une toxine (la saporine). Nous avons démontré une activité in vitro évidente de ce conjugué dans les cellules de glioblastome.Dans cette étude, nous avons utilisé d’autres agents ciblant une autre proteine surexprimée à la surface des cellules cancereuses: la nucléoline. Dans ce contexte, un aptamer et un pseudopetide se liant specifiquement à cette dernière ont été developpé et étudié.L’aptamer AS1411 (Antisoma, UK) est un agent ciblant la nucleolin approuvé par la FDA (food and drug administration). Il se lie à la nucleoline et est internalisé, perturbant ainsi l’interaction de cette dernière avec ses partenaires inhibant ainsi proliferation cellulaire.En parallèle, notre groupe a developpé un pseudopeptide, antagoniste de la nucleoline (Nucant, N6L). Il a été montré qu’il inhibe drastiquement la croissance tumorale dans le cancer du sein en induisant l’apoptose et il est actuellement en préparation pour une phase II d’essai clinique (IPP-204106). Nous avons démontré l’effet anti-proliferatif du N6L in vitro sur des cellules primaires de glioblastome.La surexpression de la nucleoline à la surface des cellules de glioblastomes couplée à la specificité de l’aptamere et du N6L pour cette deniere nous ont amené à vouloir augmenter leur efficacité d’action en les liant à une toxine. Le gène codant pour la saporine (proteine inhibitrice des ribosomes ) ou la saporine elle-meme a donc été lié à l’aptamere et au N6L.Nous avons donc dans ce travail etudié l’activité cytotoxique de l’aptamere et du N6L liés à la saporine. Les résultats obtenus sont evalués pour des futurs publications.Toutes les approches ciblées décrites, en dépit de certains problèmes, semblent prometteuses et nécessitent d'autres recherches, mais confirment que l'exploitation de cibles pour fournir des substances toxiques est l'avenir de la thérapie pour les formes cancéreuses difficiles à battre avec les thérapies conventionnelles. / Targeted cancer therapies are drugs designed to interfere with specific molecules necessary for tumor growth and progression. Traditional cytotoxic chemotherapies usually kill rapidly dividing cells in the body by interfering with cell division. A primary goal of targeted therapies is to fight cancer cells with more precision and potentially fewer side effects.Antibody-based therapy for cancer has become established over the past 15 years and is now one of the most successful and important targeted strategies. In some cases, monoclonal antibodies are conjugated to radio-isotopes or toxins (immunotoxin) to allow specific delivery of these cytotoxic agents to the intended cancer cell target. Furthermore thargeted therapies may be based also on the use of targeting molecues other than antibodies, such as peptides, growth factors, and also nucleic acids.Indeed, in this work we studyed a multi targeting strategy to deliver toxic substances (protein toxin or its gene) to cancer cells (glioblastoma).Our group published a paper describing the use of PDZ protein domain of hCASK (serine kinase calcium/calmodulin-dependent of MAGUK family) and to exploit the ability of this protein to bind to the C-terminus of hCD98 in the extracellular space. CD98 is an interesting target because it is overexpressed in different types of tumors (Giansanti F., 2015). hCASK-PDZ was genetically fused to the toxin saporin and this chimeric toxin proved to be active on glioblastoma cells in vitro.Other cell killing agents were designed to recognize and bind specifically nucleolin (NCL). This multifunctional protein is overexpressed on the surface of activated endothelial and tumor cells. In this context, compounds targeting NCL, such an aptamer, and a multivalent pseudopeptide, have been developed and investigated for cancer therapy.The aptamer against NCL, NCL-APT also known as AS1411 (Antisoma, UK), is a US Food and Drug Administration (FDA)-approved NCL targeting agent. It binds to NCL on the cell surface, preferentially gets internalized, and inhibits cancer cell growth sparing normal cells (Bates PJ, 2009).In parallel, our group, recently developed a multivalent synthetic pseudopeptide N6L that selectively binds to nucleolin (Destouches D., 2011). N6L strongly inhibits breast cancer growth by inducing apoptosis of tumor cells and is currently in preparation for phase II clinical trials (IPP-204106). We demonstrated the anti-proliferative effect of N6L on human glioblastoma cells in primary culture prepared form post-surgical specimens (Benedetti E, 2015).The overexpression of NCL on glioblastoma cell surface and the recognized selectivity of AS1411 and N6L prompted us to study a way to increase the efficiency of these ligands binding them Saporin coding gene or the protein toxin Saporin-S6, a type 1 RIP (Ribosome-Inactivating Protein) widely studied because of its potential therapeutic application in a variety of human diseases as toxic moiety of a conjugate.The characterization of the toxic activity of AS1411 linked to saporin gene (APT-SAP) and of NCL linked to saporin protein (SAP-N6L) is therefore described. Both these researches are under evaluation for publication.All the described thargeted approaches, nothwithstanding some problems, look promising and need further research, but confirm the fact that exploiting targets to deliver toxic substances is the future of therapy for cancer forms that are difficult to beat with conventional therapies.

Die Osteoarthrose (OA) des Ellbogengelenks ist eine der wichtigsten Gelenkerkrankungen des Hundes, die sich aufgrund erblich bedingter Fehlstellung der Gelenke sekundär häufig schon bei sehr jungen Hunden entwickelt und zu einer verminderten Lebensqualität der Tiere führt. Im Laufe der Erkrankung kommt es zu einer Schädigung des Gelenkknorpels, der an Elastizität und Spannkraft verliert und seine Funktion, nicht mehr ausüben kann. Eine Ablösung von Knorpelfragmenten und Entzündungen im Gelenk können die Zerstörung des Gelenkknorpels beschleunigen. Die OA ist nicht heilbar. Das Therapieziel ist eine möglichst vollständige Schmerzreduktion und die Verbesserung der Lebensqualität. Eine chirurgische Denervation ist im Ellbogengelenk des Hundes ohne Traumatisierung anatomischer Strukturen nicht möglich. Ziel der vorliegenden Arbeit war es, erstmalig einen experimentellen Ansatz zur chemischen Denervation sensibler Gelenkafferenzen im Ellbogengelenk beim Beagle mit dem Neurotoxin OX7-Saporin durchzuführen. Zusätzlich wurden Substanzen aus der Humanmedizin zur Schmerzreduktion und symptomatischen Therapie der OA am Hund erprobt und der Therapieerfolg evaluiert. Im Rahmen eines Therapieversuches in der Klinik für Kleintiere der Universität Leipzig wurde bei zwei, an OA erkrankten Hunden Capsaicin einmalig intraartikulär injiziert. Der erste Hund erhielt intraartikulär 250 mg Capsaicin. Dieser Patient zeigte Nebenwirkungen in Form einer Herz- und Atemfrequenzerhöhung, starker Schmerzhaftigkeit und neurologischen Symptomen. Der zweite Hund wurde mit 83 mg Capsaicin behandelt und zeigte keine Nebenwirkungen. Sowohl die Bewertung der Besitzer als auch die orthopädische Untersuchung ergaben keine Verbesserung der Lahmheit. Vier kranken Hunden wurde ebenfalls im Rahmen eines Therapieversuches Botox (Botulinumtoxin A) intraartikulär einmalig injiziert. Drei Hunde wurden mit 50 Einheiten, ein Hund mit 100 Einheiten Botox behandelt. Die Applikation von Capsaicin und Botox führten zu keiner Verbesserung der Symptomatik. In der Humanmedizin ist die Schmerzbehandlung mit Capsaicin und Botox erfolgreich. Deshalb sollten diese Therapieansätze auch für die Anwendung beim Hund weiter optimiert werden. Das Neurotoxin OX7-Saporin wurde in einer Dosierung von 100 µg erstmalig in das Ellbogengelenk von drei Beagle-Hündinnen appliziert, um eine sensible Denervation des Gelenkes zu erreichen. Der retrograde Transport des Ribosomen-inaktivierenden Proteins in die Perikarya der Spinalganglienzellen, die das Ellbogengelenk sensibel innervieren, sollte eine selektive Zerstörung der Neurone durch das Neurotoxin bewirken. Der retrograde Tracer Fluoro-Gold wurde, zur Kontrolle der Wirkung des Neurotoxins, nach einer Wartezeit von 15 Tagen intraartikulär in das linke und das rechte Ellbogengelenk appliziert. Fluoro-Gold kann ausschließlich von intakten Nervenfasern transportiert werden. Die Ganglien C4 - Th3 wurden bilateral zur histologischen Auswertung entnommen und in Paraffin eingebettet. Zur Bestimmung der Gesamtneuronenanzahl wurden alle Neurone in jedem dritten Schnitt eines Ganglions gezählt. Die Summe dieser Nervenzellen ergab die Gesamtneuronenanzahl eines Ganglions. Die Einlagerung von Lipofuszin führte zu einer starken Autofluoreszenz im Zytoplasma der Neuronen. Alle Schnitte wurden mit Sudan-Schwarz gefärbt, um die Autofluoreszenz von der retrograden FG-Markierung zu unterscheiden. Die FG-Markierung konnte nur in den Ganglien C6 und Th1 im Zytoplasma sehr weniger Neuronen dokumentiert werden. Der histologische Nachweis einer Neurodegeneration nach Applikation des Neurotoxins OX7-Saporin in das linke Ellbogengelenk war negativ. In den untersuchten Spinalganglien wurden intakte Nervenzellen nachgewiesen. Auch die bilaterale intraartikuläre Injektion des retrograden Tracers FG war nicht erfolgreich. Aus den Ergebnissen dieser experimentellen Ansätze wird zusammenfassend geschlussfolgert: Ø Vor der Durchführung zukünftiger experimenteller Untersuchungen beim Hund muss die optimale Konzentration und die Wartezeit für das Neurotoxins OX7-Saporin ermittelt werden. Ø Die Rezeptoren für OX7-Saporin müssen an den Nervenzellen des Hundes zweifelsfrei nachgewiesen werden, bevor das Neurotoxin im caninen Tiermodell eingesetzt werden kann. Ø Die optimale Konzentration des Fluoreszenzfarbstoffes FG für intraartikuläre Injektionen und dessen retrograde Transportgeschwindigkeit beim Hund müssen ermittelt werden, bevor dieser Tracer wieder im Tierexperiment eingesetzt werden kann. Ø FG wurde als 1,8%ige Lösung in das Ellbogengelenk beim Beagle appliziert. Diese Konzentration war wahrscheinlich zu gering, um eine retrograde Markierung der sensiblen Neurone in den entsprechenden Spinalganglienzellen nachzuweisen. Ø Die intraartikuläre Applikation des Neurotoxins OX7-Saporin führte nicht zur selektiven Neurodegeneration. Eine Aussage zur Schmerzausschaltung und symptomatischen Therapie der OA des Hundes mit OX7-Saporin ist deshalb nicht möglich.

Os neurônios do grupamento catecolaminérgico C1, localizados na porção ventrolateral do bulbo, são classicamente conhecidos por seu envolvimento no controle cardiovascular. O modelo atual propõe que os neurônios C1 são recrutados em situações que ofereçam risco de vida aos indivíduos, desencadeando respostas generalizadas e estereotipadas em defesa da homeostase. Tais respostas envolvem ajustes cardiovasculares, imunológicos, neuroendócrinos, metabólicos, termorregulatórios e respiratórios. Ferramentas anatômicas e funcionais foram utilizadas para investigar se os neurônios C1 contribuem para os ajustes respiratórios induzidos pela hipóxia e pela hipercpania. Os resultados mostram que os neurônios C1 contribuem para a aumento da ventilação induzido pela hipóxia, mas não pela hiperpania, via aumento da frequência da respiratória. Além disso, demonstramos que o aumento da frequência respiratória promovido pela ativação do grupamento C1 depende da ativação de receptores glutamatérgicos, mas não adrenérgicos, localizados na região do complexo pré-Bötzinger. / The catecholaminergic C1 neurons, located in the rostral ventrolateral portion of the medulla, are classically known by their involvement in the cardiovascular control. Recent models suggest that C1 neurons are recruited in situations of life risk, triggering generalized and stereotyped responses to homeostasis. Such responses involve cardiovascular, immunologic, neuroendocrine, metabolic, thermoregulatory and respiratory adjustments. Thus, anatomic and functional tools were used to assess the contribution of C1 neurons to the respiratory adjustments induced by hypoxia and hypercapnia. The results show that these neurons contribute to the increase of ventilation induced by hypoxia, but not by hypercapnia, via an increase of the breathing frequency. Moreover, we demonstrated that increase of breathing frequency promoted by the activation of C1 neurons depend on the activation of glutamatergic receptors, but not adrenergic, located in the pre-Bötzinge complex.

Doctor of Philosophy(PhD) / The aims of this project were to contribute to the elucidation of the role of the cholinergic system in attention and memory, two cognitive processes severely compromised in Alzheimer’s disease (AD), and to evaluate and develop tools for the functional molecular imaging of this system with a view to improving knowledge of AD and other neurological disorders. Towards the first aim, the specific anti-cholinergic toxin 192 IgG-saporin (SAP) was administered to female Sprague-Dawley rats via either an intracerebroventricular (icv) or an intracortical route and animals were tested with a vibrissal-stimulation reaction-time task and an object recognition task to evaluate their attentional and mnemonic function, respectively. The second aim was approached in two ways. Firstly, relative neuronal densities from animals with icv lesions were assessed with both ex vivo and in vitro autoradiography with the specific cholinergic radiopharmaceuticals [123I]iodobenzovesamicol (123IBVM) and 125I-A-85380, ligands for the vesicular acetylcholine transporter and the nicotinic acetylcholine receptor, respectively. Secondly, a number of in vivo and in vitro studies were performed on a novel and unique molecular imaging system (TOHR), with which it had been hoped initially to image eventually SAP-lesioned animals, with a view to measuring and ameliorating its performance characteristics and assessing its in-principle suitability for small-animal molecular imaging. The behavioural studies support a critical role for the cholinergic system in normal attentional function. Additionally, in accord with literature evidence, no significant impairment was observed in mnemonic function. It is postulated however that the results observed in the intracortically-lesioned animals support the published hypothesis that cholinergic projections to the perirhinal cortex are critical for object-recognition memory. In autoradiographic studies, SAP-lesioned animals demonstrated reduced uptake of 123IBVM in multiple regions. A reduction of nicotinic receptors was also seen in SAP-lesioned animals, a novel finding supportive of the excellent characteristics of radioiodinated I-A-85380. Examination of the performance characteristics of the TOHR support in principle its utility for targeted small-animal molecular imaging studies.

Doctor of Philosophy(PhD) / The aims of this project were to contribute to the elucidation of the role of the cholinergic system in attention and memory, two cognitive processes severely compromised in Alzheimer’s disease (AD), and to evaluate and develop tools for the functional molecular imaging of this system with a view to improving knowledge of AD and other neurological disorders. Towards the first aim, the specific anti-cholinergic toxin 192 IgG-saporin (SAP) was administered to female Sprague-Dawley rats via either an intracerebroventricular (icv) or an intracortical route and animals were tested with a vibrissal-stimulation reaction-time task and an object recognition task to evaluate their attentional and mnemonic function, respectively. The second aim was approached in two ways. Firstly, relative neuronal densities from animals with icv lesions were assessed with both ex vivo and in vitro autoradiography with the specific cholinergic radiopharmaceuticals [123I]iodobenzovesamicol (123IBVM) and 125I-A-85380, ligands for the vesicular acetylcholine transporter and the nicotinic acetylcholine receptor, respectively. Secondly, a number of in vivo and in vitro studies were performed on a novel and unique molecular imaging system (TOHR), with which it had been hoped initially to image eventually SAP-lesioned animals, with a view to measuring and ameliorating its performance characteristics and assessing its in-principle suitability for small-animal molecular imaging. The behavioural studies support a critical role for the cholinergic system in normal attentional function. Additionally, in accord with literature evidence, no significant impairment was observed in mnemonic function. It is postulated however that the results observed in the intracortically-lesioned animals support the published hypothesis that cholinergic projections to the perirhinal cortex are critical for object-recognition memory. In autoradiographic studies, SAP-lesioned animals demonstrated reduced uptake of 123IBVM in multiple regions. A reduction of nicotinic receptors was also seen in SAP-lesioned animals, a novel finding supportive of the excellent characteristics of radioiodinated I-A-85380. Examination of the performance characteristics of the TOHR support in principle its utility for targeted small-animal molecular imaging studies.

Immunotoxins (ITx) represent a new, alternate class of therapeutic agent. ITx is made when the active part of a toxin is conjugated with the binding portion of an antibody that recognizes a cancer-specific antigen. The antibody component makes ITx highly specific, as it will only bind to cells displaying the correct surface antigen. This characteristic lowers the chance of nonspecific cell damage, which causes many of the severe side effects of other chemotherapeutics. The ITx we use is a conjugate of saporin toxin. Saporin is a ribosomal inhibiting protein derived from the plant Saponaria officinales, which kills the cell by inhibiting protein synthesis. ITx enters the cancer cell by binding to the cellular marker it is specific for on the cell surface. From there, it is endocytosed, compartmentalized in an endosome, and eventually escapes to the cytosol where its ribosomal target is located. Increasing the rate of escape to the cytosol is the key to increasing cell death. The mechanism by which saporin escapes the endosome and enters the cytosol is poorly understood. Two potential mechanisms involving the rupture of the endocytic vesicle were examined. Through experiments using large unilamellar vesicles as endosomal mimics, we have been able to characterize the mechanism by which saporin works to burst the endosomal membrane through RET and calcein release. Understanding this process is the key to producing more effective immunotoxin sensitizing drugs.

O presente estudo investigou o efeito da lesão imunotóxica colinérgica específica do núcleo septal medial sobre o comportamento exploratório de ratos no labirinto em cruz elevado, em um paradigma teste-reteste, com analise minuto a minuto da primeira sessão. Uma análise bioquímica detectou uma diminuição da atividade da acetilcolinesterase no hipocampo, septo e córtex dos animais lesados com a toxina IgG-192 saporin (475 ng/µl) mas não nos animais com lesão fictícia ou nos controle. Não se encontraram diferenças significativas entre os grupos quanto aos comportamentos associados com a atividade locomotora (entradas nos braços fechados, entradas totais e distância percorrida), nem aos associados com a ansiedade (freqüência de entradas e tempo gasto nos braços abertos) nem tampouco os outros comportamentos analisados (esticar-se, levantar-se, mergulhar a cabeça e limpar-se), quando consideradas as sessões individualmente. Na comparação entre primeira e segunda sessões, os animais do grupo lesado não diminuíram a atividade exploratória durante a segunda sessão ao contrário dos animais dos grupos controle e de lesão fictícia. Um efeito semelhante foi observado nos comportamentos associados com a ansiedade e nos outros comportamentos registrados. Finalmente, uma análise minuto a minuto mostrou uma queda significativa ao longo da primeira sessão nas medidas de atividade locomotora, nos comportamentos associados com a ansiedade e nos outros comportamentos, nos animais dos grupos controle e com lesão fictícia, mas não nos animais lesados. Os resultados indicam um papel modulador da via colinérgica septo-hipocampal na atividade exploratória e nos processos de armazenamento e recuperação de informação, mas não na modulação da ansiedade, tal como avaliada no labirinto em cruz elevado. / The effect of a specific immunotoxic cholinergic lesion of the septal nucleus on exploratory behavior displayed by rats in the elevated plus maze was investigated. A five-minute test-retest approach with minute by minute analysis of the first session was used. Biochemical analyses detected a decrease in cholinesterase activity in the hippocampus, septum and cortex of animals lesioned with IgG-192 saporin (475 ng/µl) when compared with controls or sham lesioned animals. No statistical differences were found between groups in terms of behaviors associated with either locomotive activity (closed arms entries, total entries and distance percorred), anxiety (entries and time spent in the open arms) or the other behaviors recorded (stretching, rearing, head dipping and grooming). Lesioned animals did not decrease their exploratory activity in the second session as did controls and sham lesioned animals. The same was true for behaviors associated with anxiety. The minute by minute analysis showed a significant drop in exploratory as well as in anxiety associated behaviors along the first session. Results indicated a modulatory role for the cholinergic septo-hippocampal pathway in exploratory activity and its relation with storage and retrieval information processes but not for the modulation of anxiety as measured in the elevated pluz-maze.

O presente estudo investigou o efeito da lesão imunotóxica colinérgica específica do núcleo septal medial sobre o comportamento exploratório de ratos no labirinto em cruz elevado, em um paradigma teste-reteste, com analise minuto a minuto da primeira sessão. Uma análise bioquímica detectou uma diminuição da atividade da acetilcolinesterase no hipocampo, septo e córtex dos animais lesados com a toxina IgG-192 saporin (475 ng/µl) mas não nos animais com lesão fictícia ou nos controle. Não se encontraram diferenças significativas entre os grupos quanto aos comportamentos associados com a atividade locomotora (entradas nos braços fechados, entradas totais e distância percorrida), nem aos associados com a ansiedade (freqüência de entradas e tempo gasto nos braços abertos) nem tampouco os outros comportamentos analisados (esticar-se, levantar-se, mergulhar a cabeça e limpar-se), quando consideradas as sessões individualmente. Na comparação entre primeira e segunda sessões, os animais do grupo lesado não diminuíram a atividade exploratória durante a segunda sessão ao contrário dos animais dos grupos controle e de lesão fictícia. Um efeito semelhante foi observado nos comportamentos associados com a ansiedade e nos outros comportamentos registrados. Finalmente, uma análise minuto a minuto mostrou uma queda significativa ao longo da primeira sessão nas medidas de atividade locomotora, nos comportamentos associados com a ansiedade e nos outros comportamentos, nos animais dos grupos controle e com lesão fictícia, mas não nos animais lesados. Os resultados indicam um papel modulador da via colinérgica septo-hipocampal na atividade exploratória e nos processos de armazenamento e recuperação de informação, mas não na modulação da ansiedade, tal como avaliada no labirinto em cruz elevado. / The effect of a specific immunotoxic cholinergic lesion of the septal nucleus on exploratory behavior displayed by rats in the elevated plus maze was investigated. A five-minute test-retest approach with minute by minute analysis of the first session was used. Biochemical analyses detected a decrease in cholinesterase activity in the hippocampus, septum and cortex of animals lesioned with IgG-192 saporin (475 ng/µl) when compared with controls or sham lesioned animals. No statistical differences were found between groups in terms of behaviors associated with either locomotive activity (closed arms entries, total entries and distance percorred), anxiety (entries and time spent in the open arms) or the other behaviors recorded (stretching, rearing, head dipping and grooming). Lesioned animals did not decrease their exploratory activity in the second session as did controls and sham lesioned animals. The same was true for behaviors associated with anxiety. The minute by minute analysis showed a significant drop in exploratory as well as in anxiety associated behaviors along the first session. Results indicated a modulatory role for the cholinergic septo-hippocampal pathway in exploratory activity and its relation with storage and retrieval information processes but not for the modulation of anxiety as measured in the elevated pluz-maze.