L'œdème cérébral et l'hypoxie cérébrale post-traumatiques sont les acteurs principaux de l'apparition des lésions ischémiques secondaires. L'erythropoïétine (Epo) sous sa forme recombinante humaine possède une activité anti-oedémateuse dans un modèle expérimental de TC diffus. Son action sur l'hypoxie cérébrale post-traumatique reste néammoins méconnue. De plus, les effets indésirables hématologiques de l'Epo ont conduit à la synthèse de dérivés de l'Epo ne possèdant pas d'activité hématopoïétique comme l'érythropoïetine carbamylée (CEpo). Dans ce contexte, mon travail de thèse a eu pour but d'évaluer les propriétés de l'Epo et de la CEpo dans le modèle de TC diffus. Un traitement intraveineux par CEpo à la dose de 50 µg/Kg a ainsi permis de diminuer précocemment l'œdème cérébral post-traumatique évalué in vivo par IRM de diffusion et ex vivo par gravimétrie spécifique. Cette propriété a impliqué l'inhibition de la phosphorylation de la voie Erk et s'est accompagnée de l'amélioration des fonctions motrices et cognitives jusqu'à 10 jours après le TC. Après une étude de validation sur des rats sains soumis à différentes conditions d'oxygénation, une méthode de mesure IRM de la saturation locale en oxygène (lSO2) cérébrale combinant l'effet BOLD avec la mesure du volume sanguin cérébral a montré une diminution de l'oxygénation cérébrale post-traumatique. Cette hypoxie cérébrale n'était pas en lien avec une diminution du débit sanguin cérébral attestée par méthode de premier passage d'un agent de constraste. Un collapsus des capillaires cérébraux était par ailleurs retrouvé en microscopie électronique. L'Epo à la dose de 5000 UI/Kg a été capable de restaurer l'oxygénation cérébrale en diminuant l'œdème astrocytaire péricapillaire. L'ensemble de ce travail a permis d'établir les bénéfices d'un traitement par Epo ou par CEpo sur l'œdème cérébral et l'hypoxie cérébrale post-traumatiques. / Post-traumatic brain oedema and brain hypoxia play a key role for the development of secondary ischaemic lesions. Erythopoïetin (Epo) is an anti-oedematous agent in the impact-acceleration model. However its action on brain hypoxia remains unkonwn. Neuroprotective derivatives of Epo that lack haematopoïétic properties, like carbamylated Epo (CEpo), have been developped to counter Epo side effects. In this context, our study aimed to assess the effect of Epo and CEpo on post-traumatic diffuse brain oedema and brain oxygenation. CEpo (50 µg/Kg) decreased brain oedema assessed by diffusion-weighted MRI and specific gravimetry 6 hours after the trauma. The anti-oedematous effect of CEpo was linked to Erk inhibition and was associated with an improvement of cognitive and motor functions, evaluated until 10 days after the insult. MRI using the combination of BOLD contrast and blood volume fraction measurement demonstrated a decrease of local brain oxygenation in our model, without franck ischemia (measurement of mild transit time by a first passage method). Epo (5000 UI/Kg) improved brain oxygenation by decreasing post-traumatic cerebral capillaries collapse due to astrocytic end-foot swelling. All these results demonstrated that Epo and CEpo could be seen as promising neuroprotective agents in traumatic brain injury.

Abstract The goal of this study was to explore preoperative evaluation of patients with intracranial tumours using magnetic resonance imaging (MRI) methods: oedema-attenuated inversion recovery (EDAIR) sequence in low-field MRI, and diffusion-weighted imaging (DWI) and resting-state functional MRI (fMRI) in high-field MRI. The aim was also to increase our knowledge about the effects of brain surgery on eloquent brain cortices using new MRI techniques. The total number of patients in these studies was 50 (24 women). Enhancement of the tumour in ten patients after intravenous administration of gadolinium-based contrast agent in low-field MRI was examined with a new sequence, EDAIR, and compared with more conventionally used partial saturation spin echo sequences. EDAIR may facilitate the perception of small enhancing lesions and is valuable in low-field imaging, where T1-based contrast is inferior to high-field imaging. DWI was performed on 25 patients in order to evaluate the potential of this imaging method to assist in differential diagnosis of intracranial tumours. It was shown that apparent diffusion coefficient values of the tumour and peritumoural oedema produced by DWI were different in benign and malignant tumours. Resting-state blood oxygen level-dependent (BOLD) fMRI was performed on eight patients and ten healthy volunteers to examine if functional sensorimotor areas in the brain could be determined without any task-related activations. It was shown that intracranial tumours do not appear to hamper visualization of the sensorimotor area in resting-state BOLD fMRI when independent component analysis is performed, and this method may be used in preoperative imaging when activation studies cannot be performed. Conventional BOLD fMRI with motor and auditory stimuli was used with seven patients as the effect of brain surgery was studied. The results suggest that resection of a tumour with preoperative oedema probably decreases pressure on the brain and makes the functional cortex transiently more easily detectable in BOLD fMRI. In conclusion, the MRI imaging methods used in this study can give valuable additional information about the tumour, specifically for preoperative imaging and planning for surgery.

Magister Scientiae (Medical Bioscience) - MSc(MBS) / Stroke is the third leading cause of death in South Africa, killing about 240 people a day and leaving survivors with residual disabilities. There is no clinically approved neuroprotective agent for stroke at the moment but the consumption of plant polyphenols has been suggested to offer neuroprotection against stroke and other neurodegenerative diseases. In this study, we investigated the effects of long term consumption of fermented rooibos herbal tea (FRHT) on ischemia reperfusion brain injury (I-RBI) in rats. Male adult Wistar rats were fed FRHT ad libitum for 7 weeks prior to the induction of ischemic injury by the transient bilateral occlusion of the common carotid arteries (BCCAO) for 20 minutes followed by 24 hours, 4 and 7 days of reperfusion respectively. Rats were then evaluated for neurologic deficits before sacrifice and brains harvested for assessment of brain oedema, blood-brain-barrier (BBB) integrity through Evans blue extravasation (EBE), immunohistochemical studies of apoptosis and lipid peroxidation. Oxygen radical antioxidant capacity and ferric reducing antioxidant power assays were also conducted to assess total antioxidant capacity after ischemia-reperfusion injury. Notably, the long term consumption of fermented rooibos herbal tea prevented brain oedema by reducing cerebral swelling induced by I-RBI. We also observed that fermented rooibos herbal tea offered neuroprotection against damage to the BBB and delayed neuronal death associated with BCCAO as fewer apoptotic cells were identified 7 days post BCCAO reperfusion. Significantly reduced levels of lipid peroxidation and increased levels of total antioxidant capacity were also observed in brain specimens of rats treated with FRHT. Rats treated with FRHT also showed improved neurologic outcomes when compared with the untreated animals. Our results show that FRHT has potent antioxidant and anti-inflammatory properties which can provide neuroprotective effects against neuronal cell loss, cerebral swelling, BBB disruption, lipid peroxidation and neurologic deficits following I-RBI. The use of FRHT is therefore highly recommended for patients with conditions that predispose them to stroke.

No description available.

SAMMANFATTNING    Bakgrund: Venösa bensår är ett globalt vanligt förekommande fenomen, 1–2 % av jordens befolkning anses vara drabbade. Bensår skapar stort lidande för patientens fysiska och psykiska hälsa. Två vanliga förekommande problem är smärta och isolering. Det finns flera metoder för att läka venösa bensår där flerlagers-kompressionslinda anses vara en av det bästa metoderna. Att läka bensår skulle kunna minska patientlidandet.    Syfte: Att sammanställa och jämföra läkningseffekten av två- och fyra-lagerskompressionslindning hos patienter med venösa bensår.   Metod: En deskriptiv design med litteraturöversikt användes som metod där tio vetenskapliga kvantitativa originalartiklar inkluderades. Artikelsökning gjordes i databaserna PubMed och CINAHL.   Resultat: Litteraturöversikten kunde inte påvisa en skillnad mellan 2LB- och 4LB-kompressiongrupperna för förbättrad och fullständig läkningsförmåga. Båda lindorna bekräftades ha en sårläkande effekt. Effekten av kompressionslindorna gav en mer påskyndad sårläkning, än hos patienter utan kompressionsbehandling. Majoriteten av de inkluderade studierna visade på ett liknande resultat i fullständig procentuell läkning mellan två-lagerkompressionslindning (2LB) och fyra-lagerkompressionslindning (4LB).   Slutsats: Litteraturöversikten visar att kompressionsbehandling med 2LB och 4LB har en likvärdig och god sårläkande effekt för patienter. Både 2LB och 4LB är mer gynnsam för sårläkning och välmående än utan kompressionsbehandling för patienter som lever med venösa bensår. / ABSTRACT Background: Venous leg ulcers is a globally common phenomenon, 1-2 % of the world’s population is believed to be affected. Leg ulcers causes great suffering for the patient's physical and mental health. Two common complications are pain and isolation. There are several methods to heal venous leg ulcers, multilayer-compression-therapy is considered one of the best methods. Healing leg ulcers could reduce patient suffering. Purpose: To compile and compare the healing effects of two- and four-layer compression therapy in patients with venous leg ulcers.   Method: A descriptive design with a literature review as method, with ten scientific quantitative original articles were included. Article searches was done in databases PubMed and CINAHL.   Results: The results did not show a difference between the 2LB and 4LB-groups regarding complete and improved healing. However, both methods had a healing effect. The bandages were more effective in accelerating healing in patients, than in patients without compression therapy. Most of the included studies showed a similar result in the percentage of complete healing effect between the two-layer compression therapy (2LB) and four-layer compression therapy (4LB).   Conclusion: This literature review demonstrated that compression therapy with 2LB and 4LB has an equally as well as good healing effect for patients. Both 2LB and 4LB are significantly more beneficial for wound healing and well-being than without compression therapy for patients living with venous leg ulcers.

Tithonia diversifolia Hemsl. A. Gray (margaridão, Asteraceae) é uma planta medicinal com propriedade anti-inflamatória bastante promissora, embora tenha sido parcialmente investigada do ponto de vista químico e farmacológico. Neste trabalho, a partir de suas folhas foram obtidos um infuso (chá), um extrato de lavagem foliar em acetona rico em lactonas sesquiterpênicas (LSTs), um extrato em metanol-H2O das folhas livres de tricomas (ELT) e o óleo essencial, incluindo das inflorescências. Pela primeira vez os constituintes polares das folhas de T. diversifolia foram identificados e o seu mecanismo de ação anti-inflamatório foi investigado. Através dos perfis químicos dos extratos obtidos por CLAE-UV-DAD e CG-EM, comparação com dados da literatura e de uma biblioteca de padrões, juntamente com a identificação de alguns constituintes isolados, foi possível identificar mais de 20 substâncias desta planta e efetuar a caracterização química de todos seus extratos. O sucesso na obtenção de extratos que refletem diferentes classes de metabólitos secundários de T. diversifolia, avaliados por ensaios anti-inflamatórios in vivo (tópico e oral) e in vitro, permitiu concluir que as LSTs não são as únicas substâncias que contribuem para atividade anti-inflamatória da planta como se acreditava anteriormente. O extrato polar (ELT), rico em ácidos clorogênicos (ACGs) e livre de LSTs, apresentou atividade anti-inflamatória superior à do extrato contendo LSTs, do fármaco de referência (indometacina) e do fitoterápico Acheflan®. Muito similar quimicamente ao ELT, o infuso não apresentou atividade anti-inflamatória interessante, o que nos leva a desestimular seu uso popular. Ainda foi possível demonstrar que a forma de preparar cada extrato causa influência na atividade farmacológica. O estudo do óleo essencial das folhas e inflorescências permitiu observar que seus mono e sesquiterpenos também apresentam atividade anti-inflamatória. Os mecanismos de ação propostos através dos ensaios in vivo e in vitro permitiram definir que as LSTs e/ou flavonas e os ACGs de T. diversifolia são bons anti-inflamatórios, inclusive sem apresentar os efeitos colaterais comuns aos anti-inflamatórios não esteroidais atuais. Além disso, foi possível observar que a toxicidade de todos os extratos testados por via oral foi menor que aquela apresentada pelo fármaco de referência (indometacina) correntemente utilizado na terapêutica. Com base nos resultados obtidos, pode-se afirmar que a T. diversifolia brasileira possui diferentes substâncias com propriedades anti-inflamatórias que agem por diferentes mecanismos de ação. Em pelo menos um destes mecanismos, os extratos demonstraram agir sinergicamente. Embora o emprego de seu infuso não seja recomendado como anti-inflamatório, esta planta é promissora para o desenvolvimento de novos fitofármacos ou como fonte de substâncias anti-inflamatórias. / Tithonia diversifolia Hemsl. A. Gray (Mexican sunflower, Asteraceae) is a very promising anti-inflammatory medicinal plant, although so far it has been partially investigated from the chemical and pharmacological point of view. In this work, an infusion, a sesquiterpene lactone (STL)-rich leaf rinse extract in acetone, a methanol-H2O extract from trichome-free leaves (TFL), and the essential oil were obtained from the leaves of T. diversifolia, including the essential oil from its inflorescences. The main polar constituents were identified in the leaves of T. diversifolia and its anti-inflammatory mechanism of action was determined for the first time. More than 20 compounds from this plant were identified and all extracts were chemically characterized based on chemical profiles obtained from HPLC-UV-DAD and GC-MS analyzes, comparison with literature data,data from our pure compounds library, and on the identification of isolated compounds. The success in obtaining extracts with different classes of secondary metabolites of T. diversifolia allowed, after in vivo (topic and oral) and in vitro anti-inflamatory assays were carried out, to show that the STLs are not the only class of compounds that contribute to the anti-inflammatory activity of this plant as previously believed. The TFL extract, which was proved to be rich in chlorogenic acids and free of LSTs, demonstrated better activity and better mechanism of action than those displayed by the LSTs, the reference drug (indomethacin) and Acheflan® (a phytomedicine). The infusion, which is chemically very similar to the TFL extract, did not show any interesting anti-inflammatory activity, therefore discouraging its popular use. Additionally, it can be stated that the way the extracts are prepared exert influence in their pharmacological activity. The study of the essential oil allowed to observe that its mono e sesquiterpenoids also present anti-inflammatory activity. The mechanisms of action proposed after in vivo and in vitro assays were carried out allowed us to define that the STLs and/or flavones and chlorogenic acids from T. diversifolia are good anti-inflammatory agents; it should also be mentioned that these compounds do not present the side effects which are common to the current non-steroidal anti-inflammatory drugs. Furthermore, it was possible to observe that the toxicity of all extracts tested orally was lower than that presented by the reference drug (indomethacin) commonly used in the treatment of inflammatory diseases. These results altogether allowed us to conclude that the Brazilian T. diversifolia presents different classes of secondary metabolites with anti-inflammatory properties that act through different mechanisms of action. These extracts also displayed a synergistic mechanism. Although the use of the infusion is not recommended as anti-inflammatory remedy, this plant is promising for the development of new phytomedicines or as a source of anti-inflammatory compounds.

Os venenos do gênero Bothrops induzem efeitos locais caracterizados por hemorragia, necrose, edema e dor intensa. Apesar da importância clínica do fenômeno de dor, os estudos sobre os mecanismos envolvidos na gênese deste fenômeno são ainda escassos. Além disso, não existem dados sobre a capacidade do antiveneno em neutralizar este fenômeno. Neste trabalho foi investigada, a capacidade dos venenos de Bothrops jararaca, Bothrops asper e da miotoxina III (Fosfolipase A2, variante Asp 49), uma toxina isolada do veneno de Bothrops asper, em induzir hiperalgesia em ratos, a mediação química deste fenômeno e a capacidade dos antivenenos em neutralizar esta ação dos venenos. A possível correlação entre a hiperalgesia e a resposta edematogênica causada pelos venenos ou miotoxina foi também avaliada. O limiar de dor foi determinado antes e em diferentes tempos após a administração dos venenos ou toxina, empregando o teste de pressão de pata de rato. Para o estudo da resposta edematogênica, o aumento do volume das patas posteriores foi determinado por pletismografia. Os venenos e a toxina, administrados por via intraplantar, nas doses de 5µg (VBj), 15µg (VBa) ou 10µg (MIII), induziram hiperalgesia e edema, com respostas máxima na 1a (VBj, MIII) ou 2a (VBa) hora, não sendo mais detectadas na 24a hora. Para o estudo da neutralização, foram utilizados o antiveneno botrópico produzido no Instituto Butantan e o antiveneno polivalente produzido no Instituto Clodomiro Picado da Costa Rica, administrados por via endovenosa, 15 min. ou imediatamente antes ou 15 min. após a injeção dos venenos. O AVIB, quando injetado 15 min. antes do VBj, foi capaz de reverter a hiperalgesia induzida pelo veneno. Em relação ao edema, esta inibição foi observada quando o antiveneno foi administrado 15 min. ou imediatamente antes do VBj. Por outro lado, o AVCP não interferiu com a dor e o edema acarretados pelo VBa. Quando o VBj e o VBa foram incubados, in vitro, por 30 min., a 370C com os AV correspondentes, a hiperalgesia e o edema foram abolidos. Estes resultados indicam que a incapacidade do AVCP, quando administrado in vivo, de bloquear a hiperalgesia e o edema induzidos pelo VBa, não é consequência da ausência de anticorpos específicos no antiveneno, uma vez que estes efeitos foram inibidos quando o veneno foi pré-incubado com o antiveneno. Para avaliação da mediação química da hiperalgesia e do edema, os animais foram submetidos a tratamentos com inibidores de síntese, antagonistas de receptores, anticorpos ou drogas depletoras destes mediadores. Os resultados mostraram que o Hoe-140, dexametasona e NDGA inibem a hiperalgesia induzida pelo VBa, enquanto que apenas a prometazina interferiu com o edema causado pelo veneno. A hiperalgesia induzida pela MIII foi revertida pelo tratamento com prometazina, metisergida, Hoe-140, dexametasona e por NDGA, enquanto que o edema foi inibido apenas por prometazina e dexametasona. Estes dados sugerem que: a) a MIII é um importante componente do veneno para a geração de hiperalgesia, b) a bradicinina e os derivados da lipoxigenase são mediadores da dor acarretada pelo VBa e pela MIII, c) histamina e serotonina participam também da hiperalgesia induzida pela miotoxina e d) a histamina é mediador do edema induzido pelo VBa e pela MIII. Com relação à hiperalgesia induzida pelo VBj, somente o tratamento com Hoe-140 diminuiu este fenômeno, indicando a participação da bradicinina. Por outro lado, este tratamento não foi capaz de interferir com o edema induzido por este veneno. Cabe ressaltar que TEIXEIRA et al. (1994) demonstraram a participação de eicosanóides e PAF na hiperalgesia induzida pelo VBj. Os dados em conjunto sugerem ainda, dissociação entre os fenômenos de dor e edema acarretados por ambos os venenos e pela miotoxina. / Bothrops venoms cause pronounced local tissue-damage characterized by hemorrhage, myonecrosis, edema and pain. Venom-induced pain has been poorly investigated, despite its clinical relevance. Furthermore, the ability of antivenom to neutralize hyperalgesia induced by these venoms is not known. In the present study the hyperalgesia and edema induced by Bothrops jararaca (BjV) and Bothrops asper (BaV) venom and by myotoxin III-MIII (Asp49- phospholipase A2), a toxin isolated from BaV, were investigated. The chemical mediators involved in these phenomena and the ability of the antivenom to neutralize the hyperalgesia and edema induced by these venoms were also investigated. Pain threshold was assessed before and at several intervals after venom injection, using the rat paw pressure test. Edema of paw was measured phethysmographically at the same periods of time. The intraplantar injection of BjV (5µg/paw), BaV (15µg/paw) or MIII (10µg/paw) caused hyperalgesia and edema, whose peak were observed at the 1st (BjV, MIII) or 2nd (BaV) hours after venom/toxin administration, decreasing thereafter. For neutralization studies, the antivenoms produced either at Instituto Butantan from Brazil (AVIB) or Instituto Clodomiro Picado from Costa Rica (AVCP) were administered intravenously 15 min prior to, or immediately before, or 15 min after venoms injection. When the antivenom from Instituto Butantan was injected 15 min. before BjV, the hyperalgesia and edema were abolished. Furthermore, partial inhibition of edema was also observed when the antivenom was injected together with BjV. On the other hand, hyperalgesia and edema induced by BaV were not modified by AVCP. Incubation of BjV and BaV, for 30 min. at 37oC, with the antivenoms in vitro, abolished the hyperalgesia and edema. The inability of the in vivo treatment with antivenom in abolishing hyperalgesia and edema induced by BaV seems not to be related to the lack of neutralizing antibodies in antivenom, because neutralization was achieved in pre-incubation experiments. In order to investigate the chemical mediation of hyperalgesia and edema induced by the venoms or toxin, animals were treated with several drugs. Pretreatment with Hoe-140, dexamethasone and NDGA blocked the hyperalgesia induced by BaV, whereas only promethazine reduced the edema induced by this venom. The MIII-induced hyperalgesia was blocked by promethazine, methysergide, Hoe-140, dexamethasone and NDGA, whereas the edema was reduced only by promethazine and dexamethasone. These results suggest that: a) MIII may contribute to the BaV-induced hyperalgesia, b) bradykinin and leukotrienes mediate the BaV- and MIII-induced pain and MIII; c) histamine and serotonin also participate in the myotoxin-induced hyperalgesia and d) the edema induced by BaV and MIII is mediated by histamine. Pre-treatment of the animals with Hoe-140 abolished BjV-induced hyperalgesia, suggesting that bradykinin may mediate the venom-induced hyperalgesia. However, this treatment did not modify the BjV-induced edema. It is important to stress that previous studies have shown that BjV-induced hyperalgesia is mediated, at least partially, by eicosanoids and PAF (TEIXEIRA et al.,1994). The data presented herein also suggest that distinct mechanisms may be involved in the development of hyperalgesia and edema induced by both venoms and myotoxin III.

Background: Our innate immunesystem protects us from infections but, since its methods is not all specific for microorganisms, may also induce collateral damage. Severe physical injury often proved deadly throughout evolution. Such injuries may induce massive collateral damage. Nowadays we can initiate advanced critical care for affected patients and save them from imminent trauma-related death. We are therefore faced with the fact that the collateral damage from the immune system may pose a major threat to the patient, the pathophysiology of which is not amenable to direct medical treatment and which leaves us with only passive supportive measures. In this thesis we investigated the role of leucocytes under such circumstances. Our main aim was to understand better the role of leucocytes in the development of increased vascular permeability after burns and trauma. More specifically we investigated the impact of an injury on the function of leucocytes such as the dynamic change of certain cell-surface receptors on the leucocytes and in their numbers and immature forms. We wanted to find out if the increased pulmonary vascular permeability after a burn could be mediated through heparin binding protein (HBP) released from granuloctes, and whether HBP could be used as a biomarker for respiratory failure after trauma. We also wanted to confirm the possible role of histamine as a mediator of the systemic increase in vascular permeability after burns. Methods: The dynamic change of cell-surface receptors was measured by flow-acquired cytometer scanning (FACS) on blood samples taken after burns. The concentrations of HBP after a burn and mechanical trauma were analysed in plasma. Pulmonary vascular permeability after a burn was assessed using transpulmonary thermodilution. The histamine turnover after a burn was assessed with high performance liquid chromatography (HPLC) for concentrations of histamine and methylhistamine in urine. Results: We confirmed earlier investigations showing altered expression of receptors on leucocytes after a burn, receptors intimately associated with leucocyte functions (study I). In a pilot study of 10 patients we measured plasma concentrations of HBP and found them to be increased soon after a burn (study II). This finding was not confirmed in a larger, more extensive and specific study of 20 patients. We did, however, find an association between alterations in the number of leucocytes soon after a burn and pulmonary vascular permeability, indicating that they had a role in this process (study III). In another study of trauma (non burn) we found an association between the concentration of HBP in early plasma-samples after injury and the development of ARDS, indicating that granulocytes and HBP have a role in its aetiology (study IV). We found a small increase in urinary histamine and normal urinary methylhistamine concentrations but had anticipated a distinct increase followed by a decrease after reading the current papers on the subject. This indicates that the role of histamine as a mediator of increased vascular permeability after burns may have been exaggerated (study V). Conclusions: We conclude that leucocytes are affected by burns and trauma, and it is likely that they contribute to the development of respiratory failure and acute respiratory distress syndrome (ARDS). HBP is a candidate biomarker for the early detection of ARDS after trauma, and the white blood count (WBC) is a useful biomarker for the detection of decreased oxygenation soon after a burn.

Os venenos do gênero Bothrops induzem efeitos locais caracterizados por hemorragia, necrose, edema e dor intensa. Apesar da importância clínica do fenômeno de dor, os estudos sobre os mecanismos envolvidos na gênese deste fenômeno são ainda escassos. Além disso, não existem dados sobre a capacidade do antiveneno em neutralizar este fenômeno. Neste trabalho foi investigada, a capacidade dos venenos de Bothrops jararaca, Bothrops asper e da miotoxina III (Fosfolipase A2, variante Asp 49), uma toxina isolada do veneno de Bothrops asper, em induzir hiperalgesia em ratos, a mediação química deste fenômeno e a capacidade dos antivenenos em neutralizar esta ação dos venenos. A possível correlação entre a hiperalgesia e a resposta edematogênica causada pelos venenos ou miotoxina foi também avaliada. O limiar de dor foi determinado antes e em diferentes tempos após a administração dos venenos ou toxina, empregando o teste de pressão de pata de rato. Para o estudo da resposta edematogênica, o aumento do volume das patas posteriores foi determinado por pletismografia. Os venenos e a toxina, administrados por via intraplantar, nas doses de 5µg (VBj), 15µg (VBa) ou 10µg (MIII), induziram hiperalgesia e edema, com respostas máxima na 1a (VBj, MIII) ou 2a (VBa) hora, não sendo mais detectadas na 24a hora. Para o estudo da neutralização, foram utilizados o antiveneno botrópico produzido no Instituto Butantan e o antiveneno polivalente produzido no Instituto Clodomiro Picado da Costa Rica, administrados por via endovenosa, 15 min. ou imediatamente antes ou 15 min. após a injeção dos venenos. O AVIB, quando injetado 15 min. antes do VBj, foi capaz de reverter a hiperalgesia induzida pelo veneno. Em relação ao edema, esta inibição foi observada quando o antiveneno foi administrado 15 min. ou imediatamente antes do VBj. Por outro lado, o AVCP não interferiu com a dor e o edema acarretados pelo VBa. Quando o VBj e o VBa foram incubados, in vitro, por 30 min., a 370C com os AV correspondentes, a hiperalgesia e o edema foram abolidos. Estes resultados indicam que a incapacidade do AVCP, quando administrado in vivo, de bloquear a hiperalgesia e o edema induzidos pelo VBa, não é consequência da ausência de anticorpos específicos no antiveneno, uma vez que estes efeitos foram inibidos quando o veneno foi pré-incubado com o antiveneno. Para avaliação da mediação química da hiperalgesia e do edema, os animais foram submetidos a tratamentos com inibidores de síntese, antagonistas de receptores, anticorpos ou drogas depletoras destes mediadores. Os resultados mostraram que o Hoe-140, dexametasona e NDGA inibem a hiperalgesia induzida pelo VBa, enquanto que apenas a prometazina interferiu com o edema causado pelo veneno. A hiperalgesia induzida pela MIII foi revertida pelo tratamento com prometazina, metisergida, Hoe-140, dexametasona e por NDGA, enquanto que o edema foi inibido apenas por prometazina e dexametasona. Estes dados sugerem que: a) a MIII é um importante componente do veneno para a geração de hiperalgesia, b) a bradicinina e os derivados da lipoxigenase são mediadores da dor acarretada pelo VBa e pela MIII, c) histamina e serotonina participam também da hiperalgesia induzida pela miotoxina e d) a histamina é mediador do edema induzido pelo VBa e pela MIII. Com relação à hiperalgesia induzida pelo VBj, somente o tratamento com Hoe-140 diminuiu este fenômeno, indicando a participação da bradicinina. Por outro lado, este tratamento não foi capaz de interferir com o edema induzido por este veneno. Cabe ressaltar que TEIXEIRA et al. (1994) demonstraram a participação de eicosanóides e PAF na hiperalgesia induzida pelo VBj. Os dados em conjunto sugerem ainda, dissociação entre os fenômenos de dor e edema acarretados por ambos os venenos e pela miotoxina. / Bothrops venoms cause pronounced local tissue-damage characterized by hemorrhage, myonecrosis, edema and pain. Venom-induced pain has been poorly investigated, despite its clinical relevance. Furthermore, the ability of antivenom to neutralize hyperalgesia induced by these venoms is not known. In the present study the hyperalgesia and edema induced by Bothrops jararaca (BjV) and Bothrops asper (BaV) venom and by myotoxin III-MIII (Asp49- phospholipase A2), a toxin isolated from BaV, were investigated. The chemical mediators involved in these phenomena and the ability of the antivenom to neutralize the hyperalgesia and edema induced by these venoms were also investigated. Pain threshold was assessed before and at several intervals after venom injection, using the rat paw pressure test. Edema of paw was measured phethysmographically at the same periods of time. The intraplantar injection of BjV (5µg/paw), BaV (15µg/paw) or MIII (10µg/paw) caused hyperalgesia and edema, whose peak were observed at the 1st (BjV, MIII) or 2nd (BaV) hours after venom/toxin administration, decreasing thereafter. For neutralization studies, the antivenoms produced either at Instituto Butantan from Brazil (AVIB) or Instituto Clodomiro Picado from Costa Rica (AVCP) were administered intravenously 15 min prior to, or immediately before, or 15 min after venoms injection. When the antivenom from Instituto Butantan was injected 15 min. before BjV, the hyperalgesia and edema were abolished. Furthermore, partial inhibition of edema was also observed when the antivenom was injected together with BjV. On the other hand, hyperalgesia and edema induced by BaV were not modified by AVCP. Incubation of BjV and BaV, for 30 min. at 37oC, with the antivenoms in vitro, abolished the hyperalgesia and edema. The inability of the in vivo treatment with antivenom in abolishing hyperalgesia and edema induced by BaV seems not to be related to the lack of neutralizing antibodies in antivenom, because neutralization was achieved in pre-incubation experiments. In order to investigate the chemical mediation of hyperalgesia and edema induced by the venoms or toxin, animals were treated with several drugs. Pretreatment with Hoe-140, dexamethasone and NDGA blocked the hyperalgesia induced by BaV, whereas only promethazine reduced the edema induced by this venom. The MIII-induced hyperalgesia was blocked by promethazine, methysergide, Hoe-140, dexamethasone and NDGA, whereas the edema was reduced only by promethazine and dexamethasone. These results suggest that: a) MIII may contribute to the BaV-induced hyperalgesia, b) bradykinin and leukotrienes mediate the BaV- and MIII-induced pain and MIII; c) histamine and serotonin also participate in the myotoxin-induced hyperalgesia and d) the edema induced by BaV and MIII is mediated by histamine. Pre-treatment of the animals with Hoe-140 abolished BjV-induced hyperalgesia, suggesting that bradykinin may mediate the venom-induced hyperalgesia. However, this treatment did not modify the BjV-induced edema. It is important to stress that previous studies have shown that BjV-induced hyperalgesia is mediated, at least partially, by eicosanoids and PAF (TEIXEIRA et al.,1994). The data presented herein also suggest that distinct mechanisms may be involved in the development of hyperalgesia and edema induced by both venoms and myotoxin III.

Tithonia diversifolia Hemsl. A. Gray (margaridão, Asteraceae) é uma planta medicinal com propriedade anti-inflamatória bastante promissora, embora tenha sido parcialmente investigada do ponto de vista químico e farmacológico. Neste trabalho, a partir de suas folhas foram obtidos um infuso (chá), um extrato de lavagem foliar em acetona rico em lactonas sesquiterpênicas (LSTs), um extrato em metanol-H2O das folhas livres de tricomas (ELT) e o óleo essencial, incluindo das inflorescências. Pela primeira vez os constituintes polares das folhas de T. diversifolia foram identificados e o seu mecanismo de ação anti-inflamatório foi investigado. Através dos perfis químicos dos extratos obtidos por CLAE-UV-DAD e CG-EM, comparação com dados da literatura e de uma biblioteca de padrões, juntamente com a identificação de alguns constituintes isolados, foi possível identificar mais de 20 substâncias desta planta e efetuar a caracterização química de todos seus extratos. O sucesso na obtenção de extratos que refletem diferentes classes de metabólitos secundários de T. diversifolia, avaliados por ensaios anti-inflamatórios in vivo (tópico e oral) e in vitro, permitiu concluir que as LSTs não são as únicas substâncias que contribuem para atividade anti-inflamatória da planta como se acreditava anteriormente. O extrato polar (ELT), rico em ácidos clorogênicos (ACGs) e livre de LSTs, apresentou atividade anti-inflamatória superior à do extrato contendo LSTs, do fármaco de referência (indometacina) e do fitoterápico Acheflan®. Muito similar quimicamente ao ELT, o infuso não apresentou atividade anti-inflamatória interessante, o que nos leva a desestimular seu uso popular. Ainda foi possível demonstrar que a forma de preparar cada extrato causa influência na atividade farmacológica. O estudo do óleo essencial das folhas e inflorescências permitiu observar que seus mono e sesquiterpenos também apresentam atividade anti-inflamatória. Os mecanismos de ação propostos através dos ensaios in vivo e in vitro permitiram definir que as LSTs e/ou flavonas e os ACGs de T. diversifolia são bons anti-inflamatórios, inclusive sem apresentar os efeitos colaterais comuns aos anti-inflamatórios não esteroidais atuais. Além disso, foi possível observar que a toxicidade de todos os extratos testados por via oral foi menor que aquela apresentada pelo fármaco de referência (indometacina) correntemente utilizado na terapêutica. Com base nos resultados obtidos, pode-se afirmar que a T. diversifolia brasileira possui diferentes substâncias com propriedades anti-inflamatórias que agem por diferentes mecanismos de ação. Em pelo menos um destes mecanismos, os extratos demonstraram agir sinergicamente. Embora o emprego de seu infuso não seja recomendado como anti-inflamatório, esta planta é promissora para o desenvolvimento de novos fitofármacos ou como fonte de substâncias anti-inflamatórias. / Tithonia diversifolia Hemsl. A. Gray (Mexican sunflower, Asteraceae) is a very promising anti-inflammatory medicinal plant, although so far it has been partially investigated from the chemical and pharmacological point of view. In this work, an infusion, a sesquiterpene lactone (STL)-rich leaf rinse extract in acetone, a methanol-H2O extract from trichome-free leaves (TFL), and the essential oil were obtained from the leaves of T. diversifolia, including the essential oil from its inflorescences. The main polar constituents were identified in the leaves of T. diversifolia and its anti-inflammatory mechanism of action was determined for the first time. More than 20 compounds from this plant were identified and all extracts were chemically characterized based on chemical profiles obtained from HPLC-UV-DAD and GC-MS analyzes, comparison with literature data,data from our pure compounds library, and on the identification of isolated compounds. The success in obtaining extracts with different classes of secondary metabolites of T. diversifolia allowed, after in vivo (topic and oral) and in vitro anti-inflamatory assays were carried out, to show that the STLs are not the only class of compounds that contribute to the anti-inflammatory activity of this plant as previously believed. The TFL extract, which was proved to be rich in chlorogenic acids and free of LSTs, demonstrated better activity and better mechanism of action than those displayed by the LSTs, the reference drug (indomethacin) and Acheflan® (a phytomedicine). The infusion, which is chemically very similar to the TFL extract, did not show any interesting anti-inflammatory activity, therefore discouraging its popular use. Additionally, it can be stated that the way the extracts are prepared exert influence in their pharmacological activity. The study of the essential oil allowed to observe that its mono e sesquiterpenoids also present anti-inflammatory activity. The mechanisms of action proposed after in vivo and in vitro assays were carried out allowed us to define that the STLs and/or flavones and chlorogenic acids from T. diversifolia are good anti-inflammatory agents; it should also be mentioned that these compounds do not present the side effects which are common to the current non-steroidal anti-inflammatory drugs. Furthermore, it was possible to observe that the toxicity of all extracts tested orally was lower than that presented by the reference drug (indomethacin) commonly used in the treatment of inflammatory diseases. These results altogether allowed us to conclude that the Brazilian T. diversifolia presents different classes of secondary metabolites with anti-inflammatory properties that act through different mechanisms of action. These extracts also displayed a synergistic mechanism. Although the use of the infusion is not recommended as anti-inflammatory remedy, this plant is promising for the development of new phytomedicines or as a source of anti-inflammatory compounds.