RESUMO
Following stroke, the survival of neurons and their ability to reestablish connections is critical to functional recovery. This is strongly influenced by the balance between neuronal excitation and inhibition. In the acute phase of experimental stroke, lethal hyperexcitability can be attenuated by positive allosteric modulation of GABAA receptors (GABAARs). Conversely, in the late phase, negative allosteric modulation of GABAAR can correct the suboptimal excitability and improves both sensory and motor recovery. Here, we hypothesized that octadecaneuropeptide (ODN), an endogenous allosteric modulator of the GABAAR synthesized by astrocytes, influences the outcome of ischemic brain tissue and subsequent functional recovery. We show that ODN boosts the excitability of cortical neurons, which makes it deleterious in the acute phase of stroke. However, if delivered after day 3, ODN is safe and improves motor recovery over the following month in two different paradigms of experimental stroke in mice. Furthermore, we bring evidence that, during the subacute period after stroke, the repairing cortex can be treated with ODN by means of a single hydrogel deposit into the stroke cavity.SIGNIFICANCE STATEMENT Stroke remains a devastating clinical challenge because there is no efficient therapy to either minimize neuronal death with neuroprotective drugs or to enhance spontaneous recovery with neurorepair drugs. Around the brain damage, the peri-infarct cortex can be viewed as a reservoir of plasticity. However, the potential of wiring new circuits in these areas is restrained by a chronic excess of GABAergic inhibition. Here we show that an astrocyte-derived peptide, can be used as a delayed treatment, to safely correct cortical excitability and facilitate sensorimotor recovery after stroke.
Assuntos
Inibidor da Ligação a Diazepam/uso terapêutico , Agonistas de Receptores de GABA-A/uso terapêutico , Neurônios/efeitos dos fármacos , Neuropeptídeos/uso terapêutico , Fragmentos de Peptídeos/uso terapêutico , Receptores de GABA-A/efeitos dos fármacos , Acidente Vascular Cerebral/tratamento farmacológico , Adulto , Animais , Astrócitos/metabolismo , Depressão Alastrante da Atividade Elétrica Cortical/fisiologia , Inibidor da Ligação a Diazepam/deficiência , Inibidor da Ligação a Diazepam/fisiologia , Implantes de Medicamento , Potenciais Somatossensoriais Evocados , Feminino , Agonistas de Receptores de GABA-A/farmacologia , Humanos , Hidrogéis , Infarto da Artéria Cerebral Média/tratamento farmacológico , Trombose Intracraniana/tratamento farmacológico , Trombose Intracraniana/etiologia , Luz , Camundongos , Camundongos Endogâmicos C57BL , N-Metilaspartato/toxicidade , Neurônios/fisiologia , Neuropeptídeos/deficiência , Neuropeptídeos/fisiologia , Técnicas de Patch-Clamp , Fragmentos de Peptídeos/deficiência , Fragmentos de Peptídeos/fisiologia , Ratos , Rosa Bengala/efeitos da radiação , Rosa Bengala/toxicidade , Método Simples-Cego , Acidente Vascular Cerebral/etiologiaRESUMO
Pancreatic cancer remains one of the most lethal forms of cancer with a 10-year survival of <1%. With little improvement in survival rates observed in the past 40â¯years, there is a significant need for new treatments or more effective strategies to deliver existing treatments. The antimetabolite gemcitabine (Gem) is the most widely used form of chemotherapy for pancreatic cancer treatment, but is known to produce significant side effects when administered systemically. We have previously demonstrated the benefit of combined chemo-sonodynamic therapy (SDT), delivered using oxygen carrying microbubbles (O2MB), as a targeted treatment for pancreatic cancer in a murine model of the disease. In this manuscript, we report the preparation of a biotin functionalised Gem ligand for attachment to O2MBs (O2MB-Gem). We demonstrate the effectiveness of chemo-sonodynamic therapy following ultrasound-targeted-microbubble-destruction (UTMD) of the O2MB-Gem and a Rose Bengal loaded O2MB (O2MB-RB) as a targeted treatment for pancreatic cancer. Specifically, UTMD using the O2MB-Gem and O2MB-RB conjugates reduced the viability of MIA PaCa-2, PANC-1, BxPC3 and T110299 pancreatic cancer cells by >60% (pâ¯<â¯0.001) and provided significant tumour growth delay (>80%, pâ¯<â¯0.001) compared to untreated animals when human xenograft MIA PaCa-2 tumours were treated in SCID mice. The toxicity of the O2MB-Gem conjugate was also determined in healthy non-tumour bearing MF1 mice and revealed no evidence of renal or hepatic damage. Therefore, the results presented in this manuscript suggest that chemo-sonodynamic therapy using the O2MB-Gem and O2MB-RB conjugates, is potentially an effective targeted and safe treatment modality for pancreatic cancer.
Assuntos
Antimetabólitos Antineoplásicos/administração & dosagem , Desoxicitidina/análogos & derivados , Microbolhas , Neoplasias Pancreáticas/tratamento farmacológico , Animais , Antimetabólitos Antineoplásicos/farmacologia , Antimetabólitos Antineoplásicos/toxicidade , Linhagem Celular Tumoral , Desoxicitidina/administração & dosagem , Desoxicitidina/farmacologia , Sistemas de Liberação de Medicamentos , Feminino , Humanos , Masculino , Camundongos SCID , Neoplasias Pancreáticas/patologia , Rosa Bengala/química , Rosa Bengala/toxicidade , Terapia por Ultrassom/métodos , Ensaios Antitumorais Modelo de Xenoenxerto , GencitabinaRESUMO
We report here, reverse micelle mediated synthesis of multifunctional dextran (dex) coated Gd2O3 nanoparticles (NPs) carrying rose bengal (RB) dye for magnetic resonance and optical imaging. The diameter of these RB attached dex coated Gd2O3 NPs (Gd-dex-RB NPs) was found to be ~17â¯nm as measured by TEM. NMR line broadening effect on the surrounding water protons affirmed the paramagnetic nature of these NPs. Optical properties of Gd-dex-RB NPs were validated by UV-Vis and fluorescence spectroscopy. Time dependent release profile of RB from NPs at two different pH of 7.4 and 5.0 revealed that these NPs behave as slow releasing system. In-vitro study revealed that NPs are efficiently taken up by cells and show optical activity in cellular environment. In vitro cell viability (SRB) assay was performed on cancerous (A-549, U-87) and normal (HEK-293) cell lines, showed the absence of cytotoxic effect of Gd-dex-RB NPs. Therefore, such multifunctional NPs can be efficiently used for bio-imaging and optical tracking.
Assuntos
Meios de Contraste/química , Dextranos/química , Gadolínio/química , Imageamento por Ressonância Magnética/métodos , Nanopartículas Metálicas , Imagem Molecular/métodos , Rosa Bengala/química , Células A549 , Sobrevivência Celular/efeitos dos fármacos , Meios de Contraste/administração & dosagem , Meios de Contraste/metabolismo , Meios de Contraste/toxicidade , Dextranos/administração & dosagem , Dextranos/metabolismo , Dextranos/toxicidade , Gadolínio/administração & dosagem , Gadolínio/metabolismo , Gadolínio/toxicidade , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Cinética , Tamanho da Partícula , Rosa Bengala/administração & dosagem , Rosa Bengala/metabolismo , Rosa Bengala/toxicidade , Solubilidade , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , Água/químicaRESUMO
Rose Bengal (RB) is an anionic water-soluble xanthene dye, which used for many years to assess eye cornea and conjunctiva damage. RB showed strong absorption maxima (λmax) under visible light followed by UV-B and UV-A. RB under sunlight exposure showed a time-dependent photodegradation. Our results show that photosensitized RB generates (1)O2 via Type-II photodynamic pathway and induced DNA damage under sunlight/UV-R exposure. 2'dGuO degradation, micronuclei formation, and single- and double-strand breakage were the outcome of photogenotoxicity caused by RB. Quenching studies with NaN3 advocate the involvement of (1)O2 in RB photogenotoxicity. RB induced linoleic acid photoperoxidation, which was parallel to (1)O2-mediated DNA damage. Oxidative stress in A375 cell line (human melanoma cell line) was detected through DCF-DA assay. Photosensitized RB decreased maximum cellular viability under sunlight followed by UV-B and UV-A exposures. Apoptosis was detected as a pattern of cell death through the increased of caspase-3 activity, decreased mitochondrial membrane potential, and PS translocation through inner to outer plasma membrane. Increased cytosolic levels of Bax also advocate the apoptotic cell death. We propose a p53-mediated apoptosis via increased expression of Bax gene and protein. Thus, the exact mechanism behind RB phototoxicity was the involvement of (1)O2, which induced oxidative stress-mediated DNA and membrane damage, finally apoptotic cell death under natural sunlight exposure. The study suggests that after the use of RB, sunlight exposure may avoid to prevent from its harmful effects.
Assuntos
Melanoma/patologia , Rosa Bengala/química , Rosa Bengala/toxicidade , Luz Solar , Caspase 3/metabolismo , Linhagem Celular Tumoral , Dano ao DNA , Humanos , Ácido Linoleico/química , Melanoma/metabolismo , Potencial da Membrana Mitocondrial , Microscopia Eletrônica de Transmissão , Oxirredução , Dímeros de Pirimidina/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Oxidative damage to DNA has many origins, including irradiation, inflammation, and oxidative stress, but the chemistries are not the same. The most oxidizable base in DNA is 2-deoxyguanosine (dG), and the primary oxidation products are 8-oxodG and 2-amino-imidazolone. The latter rapidly converts to 2,2-diamino-oxazolone (Ox), and 8-oxodG is further oxidized to spiroiminodihydantoin (Sp) and guanidinohydantoin (Gh). In this study, we have examined the dose-response relationship for the formation of the above four products arising in calf thymus DNA exposed to gamma irradiation, photoactivated rose bengal, and two sources of peroxynitrite. In order to carry out these experiments, we developed a chromatographic system and synthesized isotopomeric internal standards to enable accurate and precise analysis based upon selected reaction monitoring mass spectrometry. 8-OxodG was the most abundant products in all cases, but its accumulation was highly dependent on the nature of the oxidizing agent and the subsequent conversion to Sp and Gh. Among the other oxidation products, Ox was the most abundant, and Sp was formed in significantly greater yield than Gh.
Assuntos
DNA/química , Guanina/química , Oxidantes/química , Ácido Peroxinitroso/química , Oxigênio Singlete/química , 8-Hidroxi-2'-Desoxiguanosina , Animais , Bovinos , Desoxiguanosina/análogos & derivados , Desoxiguanosina/química , Raios gama , Guanidinas/química , Guanosina/análogos & derivados , Guanosina/química , Hidantoínas/química , Oxidantes/toxicidade , Oxirredução , Ácido Peroxinitroso/toxicidade , Rosa Bengala/química , Rosa Bengala/toxicidade , Oxigênio Singlete/toxicidade , Compostos de Espiro/químicaRESUMO
OBJECTIVE: To correlate potential inflammatory responses in nonarteritic anterior ischemic optic neuropathy (NAION) with a lesion possessing many physiologic and histologic similarities from a model of nonhuman primate NAION (pNAION). METHODS: Using immunohistochemistry and confocal microscopic analysis, we evaluated the relative numbers of inflammatory cell types in the single available clinical specimen of early NAION (21 days after event). We correlated this with the temporal inflammatory response occurring in optic nerve tissue at different times following pNAION induction. RESULTS: In pNAION, there is a previously unsuspected infiltration of polymorphonuclear leukocytes occurring almost immediately after infarct induction, followed by invasion of ED1+ extrinsic macrophages, which peaks 5 weeks after infarct. Intrinsic microglia accumulate up to 70 days after induction in the area of primary axonal loss. The analyzed human NAION specimen was similar to 21-day pNAION tissue, with extrinsic macrophages and intrinsic microglial cells in the region of focal axon loss. CONCLUSIONS: Cellular inflammation plays a major early role following white-matter (optic nerve) infarct, with both polymorphonuclear leukocyte and macrophage function involved in debris elimination and tissue remodeling. The optic nerve in NAION and its primate model are associated with early cellular inflammation, previously unsuspected, that may contribute to postinfarct optic nerve damage.
Assuntos
Modelos Animais de Doenças , Macrófagos/patologia , Neutrófilos/patologia , Nervo Óptico/patologia , Neurite Óptica/imunologia , Neuropatia Óptica Isquêmica/imunologia , Idoso , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Axônios/metabolismo , Axônios/patologia , Biomarcadores/metabolismo , Proteínas de Ligação ao Cálcio , Contagem de Células , Estudos Transversais , Proteínas de Ligação a DNA/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Arterite de Células Gigantes/imunologia , Arterite de Células Gigantes/patologia , Humanos , Inflamação/metabolismo , Inflamação/patologia , Macaca mulatta , Macrófagos/metabolismo , Masculino , Proteínas dos Microfilamentos , Microscopia Confocal , Neutrófilos/metabolismo , Nervo Óptico/metabolismo , Neurite Óptica/patologia , Neuropatia Óptica Isquêmica/patologia , Rosa Bengala/toxicidadeRESUMO
As stem cells can regenerate damaged tissue, their therapeutic potential on brain damage has been investigated. In this study, the effects of embryonic stem cell transplantation on brain damage were investigated by using a photochemically induced thrombotic brain damage model. Mice with systemic transplantation of embryonic stem cells expressing enhanced green fluorescence protein on day 1 showed a smaller brain lesion size on day 8 than the control mice. The smaller lesion was accompanied by the increase in the number of microvessels at the border of the damaged area. Inside and around the damaged lesion, no EGFP-positive cells were observed. These findings suggested that embryonic stem cell transplantation reduced the brain lesion through the acceleration of angiogenesis by endogenous endothelial cells.
Assuntos
Infarto Encefálico/cirurgia , Células-Tronco Embrionárias/transplante , Trombose Intracraniana/cirurgia , Neovascularização Fisiológica/fisiologia , Regeneração Nervosa/fisiologia , Transplante de Células-Tronco/métodos , Animais , Edema Encefálico/etiologia , Edema Encefálico/fisiopatologia , Edema Encefálico/cirurgia , Infarto Encefálico/patologia , Infarto Encefálico/fisiopatologia , Capilares/citologia , Capilares/fisiologia , Técnicas de Cultura de Células , Linhagem Celular , Modelos Animais de Doenças , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/fisiologia , Corantes Fluorescentes/efeitos da radiação , Corantes Fluorescentes/toxicidade , Trombose Intracraniana/patologia , Trombose Intracraniana/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microcirculação/fisiologia , Neoplasias/etiologia , Neoplasias/patologia , Neoplasias/fisiopatologia , Estimulação Luminosa/efeitos adversos , Complicações Pós-Operatórias/etiologia , Complicações Pós-Operatórias/patologia , Complicações Pós-Operatórias/fisiopatologia , Medição de Risco , Rosa Bengala/efeitos da radiação , Rosa Bengala/toxicidade , Transplante de Células-Tronco/efeitos adversos , Transplante Homólogo/efeitos adversos , Transplante Homólogo/métodosRESUMO
The synthetic food dyes studied were rose bengal (RB), phroxine (PL), amaranth, erythrosine B (ET), allura red, new coccine, acid red (AR), tartrazine, sunset yellow FCF, brilliant blue FCF, and indigo carmine. First, data confirmed that these dyes were not substrates for CYP2A6, UGT1A6, and UGT2B7. ET inhibited UGT1A6 (glucuronidation of p-nitrophenol) and UGT2B7 (glucuronidation of androsterone). We showed the inhibitory effect of xanthene dye on human UGT1A6 activity. Basic ET, PL, and RB in those food dyes strongly inhibited UGT1A6 activity, with IC(50) values = 0.05, 0.04, and 0.015 mM, respectively. Meanwhile, AR of an acidic xanthene food dye showed no inhibition. Next, we studied the inhibition of CYP3A4 of a major phase I drug-metabolizing enzyme and P-glycoprotein of a major transporter by synthetic food dyes. Human CYP3A4 and P-glycoprotein were also inhibited by basic xanthene food dyes. The IC(50) values of these dyes to inhibit CYP3A4 and P-glycoprotein were the same as the inhibition level of UGT1A6 by three halogenated xanthene food dyes (ET, PL, and RB) described above, except AR, like the results with UGT1A6 and UGT2B7. We also confirmed the noninhibition of CYP3A4 and P-gp by other synthetic food dyes. Part of this inhibition depended upon the reaction of (1)O(2) originating on xanthene dyes by light irradiation, because inhibition was prevented by (1)O(2) quenchers. We studied the influence of superoxide dismutase and catalase on this inhibition by dyes and we found prevention of inhibition by superoxide dismutase but not catalase. This result suggests that superoxide anions, originating on dyes by light irradiation, must attack drug-metabolizing enzymes. It is possible that red cosmetics containing phloxine, erythrosine, or rose bengal react with proteins on skin under lighting and may lead to rough skin.
Assuntos
Inibidores das Enzimas do Citocromo P-450 , Inibidores Enzimáticos/toxicidade , Corantes de Alimentos/toxicidade , Glucuronosiltransferase/antagonistas & inibidores , Xantenos/toxicidade , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Corante Amaranto/toxicidade , Hidrocarboneto de Aril Hidroxilases/antagonistas & inibidores , Compostos Azo/toxicidade , Benzenossulfonatos/toxicidade , Transporte Biológico/efeitos dos fármacos , Catalase/farmacologia , Citocromo P-450 CYP2A6 , Citocromo P-450 CYP3A , Inibidores do Citocromo P-450 CYP3A , Eritrosina/toxicidade , Fluoresceínas/toxicidade , Humanos , Índigo Carmim/toxicidade , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Naftalenossulfonatos , Rodaminas/toxicidade , Rosa Bengala/toxicidade , Superóxido Dismutase/farmacologia , Tartrazina/toxicidadeRESUMO
Nonarteritic anterior ischemic optic neuropathy (NAION) results from isolated anterior optic nerve (ON)-axonal ischemia near the retina-optic nerve junction. We utilized a rodent model of NAION (rAION) to study the in vivo inflammatory response after pure axonal ischemic infarct. ON ischemia was generated using laser-coupled rose Bengal dye photoactivation, and the infarct localized using tetrazolium red and histology. ON inflammation was evaluated following infarct using extrinsic macrophage (ED1) and microglial (isolated Iba1) cell markers. In naive ONs, some ED1(+)/Iba1(+) cells, representing extrinsic macrophages, were present in intraretinal ON region, but not in the retroscleral (isolated ON) region. Numerous ED1(-)/Iba1(+) cells, likely representing intrinsic microglia, were present throughout the entire ON. One day post-stroke, slight increases in both ED1(+) and Iba1(+) cells were apparent in the eye region immediately surrounding the anterior ON. Three days post-stroke, there was marked infiltration and aggregates of ED1(+)/Iba1(+) cells, with axon structural disruption in the region of the ischemic infarct. ED1(+) and Iba1(+) cells were present in the portion of the ON surrounding the infarct, possibly representing a penumbral region similar to that seen in ischemic brain infarcts. Although ED1(+) cells decreased by 7-14 days post-stroke, large numbers of Iba1(+) cells persisted in the anterior ON. Similar to other CNS ischemic strokes, pure axonal ischemia results in the early recruitment of extrinsic macrophages to the ischemic region. Manipulation of the inflammatory response may be an important variable that could potentially improve visual outcome.
Assuntos
Nervo Óptico/patologia , Neuropatia Óptica Isquêmica/patologia , Retina/patologia , Animais , Axônios/imunologia , Axônios/patologia , Contagem de Células , Imunofluorescência , Inflamação/patologia , Macrófagos/patologia , Microglia/patologia , Nervo Óptico/irrigação sanguínea , Nervo Óptico/imunologia , Neuropatia Óptica Isquêmica/induzido quimicamente , Neuropatia Óptica Isquêmica/imunologia , Ratos , Ratos Sprague-Dawley , Retina/imunologia , Rosa Bengala/toxicidade , Fatores de TempoRESUMO
Rose Bengal (RB) is a very efficient photosensitizer which undergoes inactivation of its photophysical and photochemical properties upon addition of a quencher group-i.e. acetate-to the xanthene rings. The resulting RB acetate (RB-Ac) derivative behaves as a fluorogenic substrate: it easily enters the cells where the native photoactive molecule is restored by esterase activities. It is known that the viability of RB-Ac-loaded cells is strongly reduced by light irradiation, attesting to the formation of intracellular RB. The aim of this study was to identify the organelles photodamaged by the intracellularly formed RB. RB-Ac preloaded rat C6 glioma cells and human HeLa cells were irradiated at 530 nm. Fluorescence confocal imaging and colocalization with specific dyes showed that the restored RB molecules redistribute dynamically through the cytoplasm, with the achievement of a dynamic equilibrium at 30 min after the administration, in the cell systems used; this accounted for a generalized damage to several organelles and cell structures (i.e. the endoplasmic reticulum, the Golgi apparatus, the mitochondria, and the cytoskeleton). The multiple organelle damage, furthermore, led preferentially to apoptosis as demonstrated by light and electron microscopy and by dual-fluorescence staining with FITC-labelled annexin V and propidium iodide.
Assuntos
Apoptose , Corantes Fluorescentes/toxicidade , Fármacos Fotossensibilizantes/toxicidade , Rosa Bengala/análogos & derivados , Animais , Células HeLa , Humanos , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Organelas/efeitos dos fármacos , Ratos , Rosa Bengala/análise , Rosa Bengala/toxicidade , Raios UltravioletaRESUMO
PURPOSE: Retinal neovascularization (NV) is associated with various disorders, such as retinal vein occlusion, diabetic retinopathy, and retinopathy of prematurity, and often causes severe loss of vision. To determine the mechanism of retinal NV and develop new therapy, we developed a mouse model using a photodynamic method. METHODS: C57BL/6 mice were injected with rose bengal via the tail vein, and then selected venous points were photocoagulated. RESULTS: All eyes demonstrated venous occlusion on day 1, and capillary nonperfusion areas were observed until day 3. Twenty of 33 eyes (60.6%) developed retinal NV on day 14, confirmed by fluorescein isothiocyanate-perfused retinal flat-mounts and immunochemical and histopathological analyses. Reverse transcriptase-polymerase chain reaction showed an increase in the expression of vascular endothelial growth factor at the retina on day 7. CONCLUSIONS: Because of the simplicity, low cost, and feasibility of genetic manipulations, our model is believed to represent an advance in investigating molecular mechanisms and establishing therapy for retinal NV.
Assuntos
Modelos Animais de Doenças , Neovascularização Retiniana/patologia , Oclusão da Veia Retiniana/patologia , Animais , Dextranos , Progressão da Doença , Feminino , Fluoresceína-5-Isotiocianato/análogos & derivados , Corantes Fluorescentes/administração & dosagem , Corantes Fluorescentes/toxicidade , Seguimentos , Expressão Gênica , Injeções Intravenosas , Fotocoagulação a Laser/efeitos adversos , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , RNA/genética , Retina/metabolismo , Retina/ultraestrutura , Neovascularização Retiniana/etiologia , Neovascularização Retiniana/genética , Oclusão da Veia Retiniana/etiologia , Oclusão da Veia Retiniana/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rosa Bengala/administração & dosagem , Rosa Bengala/toxicidade , Fator A de Crescimento do Endotélio Vascular/biossíntese , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Stroke is a major cause of epilepsy, but the molecular mechanisms underlying post-stroke epileptogenesis are unknown. The expression of cystatin C, a cysteine protease inhibitor, is increased in the hippocampus during status epilepticus (SE)-induced epileptogenesis, and regulates both cell death and birth. To test the hypothesis that increased cystatin C expression represents a common molecular alteration induced by epileptogenic brain insults, we investigated the time course, cellular localization, and association of cystatin C expression with neuronal damage during post-stroke epileptogenesis. Stroke was induced with photothrombosis, which leads to epilepsy in approximately 20-30% of rats. Cystatin C expression was increased in the CA1 area of the hippocampus 4 days after photothrombosis, when the diameter of the lesion was the largest. Double-labeling and confocal analysis indicated that cystatin C was expressed in astrocytes and microglia. Unlike after SE, cystatin C expression did not change in the dentate gyrus. Also, increased cystatin C expression was not associated with neurodegeneration, which was demonstrated as an absence of Fluoro Jade B-positive cells in adjacent sections. The present study provides evidence that cystatin C may be involved in cellular alterations that occur after an epileptogenic insult, not only after SE but also after photothrombotic stroke.
Assuntos
Cistatinas/biossíntese , Hipocampo/metabolismo , Estado Epiléptico/fisiopatologia , Animais , Cistatina C , Corantes Fluorescentes/toxicidade , Hipocampo/patologia , Hipocampo/fisiopatologia , Masculino , Microscopia Confocal , Degeneração Neural/patologia , Neuroglia/metabolismo , Ratos , Ratos Sprague-Dawley , Rosa Bengala/toxicidade , Estado Epiléptico/etiologia , Estado Epiléptico/patologia , Acidente Vascular Cerebral/complicações , Acidente Vascular Cerebral/patologia , Acidente Vascular Cerebral/fisiopatologiaRESUMO
Many aspects of cellular function or physiology can be used to indicate the level of damage resulting from the application of potentially deleterious agents such as drugs, solvents or even light. The dose required to reach a specific biological endpoint will necessarily depend on the characteristics of the damage induced by the agent. By using multiple biological probes, it is possible to get a more complete description of the type of damage induced. Photodamage was induced in rat basophilic leukemia cells by either 254-nm UVC light exposure or rose bengal photosensitization. Damage was measured by three quantitative assays employing fluorescent probes: calcein, to measure nonspecific esterase activity, propidium iodide (PI), to measure loss of plasma membrane integrity, rhodamine 123 (R123) to measure mitochondrial depolarization, and the incorporation of 5'-bromodeoxyuridine (BrdU), to measure the progress of cell replication. BrdU incorporation was found to be the most sensitive indicator for both forms of photodamage. For UVC photodamage, the BrdU assay was 330 times more sensitive than the other two assays. For rose bengal photosensitization, the BrdU assay was 48 or 62 times more sensitive than either the R123 or calcein/PI assays, respectively.
Assuntos
Divisão Celular/efeitos da radiação , Leucemia/patologia , Rosa Bengala/toxicidade , Raios Ultravioleta/efeitos adversos , Animais , Bromodesoxiuridina/metabolismo , Divisão Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Esterases/metabolismo , Fluoresceínas/análise , Corantes Fluorescentes , Mitocôndrias/metabolismo , Fármacos Fotossensibilizantes/toxicidade , Propídio/química , Ratos , Rodamina 123/química , Células Tumorais CultivadasRESUMO
OBJECTIVES: The main focus of this research was to investigate the photodynamic therapy (PDT), in vitro, acting on Streptococcus mutans and fibroblasts. A hand held photopolymerizer (HHP) and a classical photosensitizer (Rose Bengal) were used to induce photodynamic response. METHODS: S. mutans and fibroblast were treated with different concentrations of Rose Bengal (0-50 microM) irradiated with light (400-500 nm) for different time periods (0-40s) and then cell viability was evaluated. RESULTS: It was observed that the light (per se) is not toxic and in the dark Rose Bengal is toxic to the cells tested only at concentrations above 2.5 microM. Under light exposure concentrations of Rose Bengal above 0.5 microM all S. mutans were killed with no cytotoxic effects to fibroblasts. CONCLUSIONS: For the purpose of this work, the photoactivation of Rose Bengal, using the HHP, inactivated the bacteria without affecting the fibroblast viability.
Assuntos
Luz , Fotoquimioterapia , Fármacos Fotossensibilizantes/toxicidade , Rosa Bengala/toxicidade , Streptococcus mutans/efeitos da radiação , Técnicas Bacteriológicas , Técnicas de Cultura de Células , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Relação Dose-Resposta a Droga , Fibroblastos/efeitos dos fármacos , Fibroblastos/efeitos da radiação , Humanos , Fotoquimioterapia/instrumentação , Streptococcus mutans/efeitos dos fármacos , Fatores de TempoRESUMO
This study characterizes by functional and electrophysiological methods changes following photochemically induced injuries to the spinal cord in adult rats. The spinal cord was exposed by laminectomy and bathed with 1.5% rose bengal solution for 10 min (T12-L1 vertebrae). The excess dye was removed by saline rinse and the spinal cord was irradiated with "cold" light for 0, 1, 2.5, 5, and 10 min in different groups of rats. During the first 15 days postlesion, locomotion activity, pain sensibility, motor and somatosensory evoked potentials, and motor and nerve action potentials were evaluated. Graded locomotor and nociceptive recovery was observed in irradiated rats depending on the photoinduction time. At 15 days, the amplitude of motor and sensory evoked potentials was significantly lower in irradiated groups with respect to control rats. The amplitude of compound muscle action potentials and of reflex H wave after sciatic nerve stimulation decreased significantly in irradiated animals with respect to control rats, while the latency did not show significant differences. In irradiated groups, significant differences were seen between pre- and postoperative values for most functional and electrophysiological parameters analyzed. A significant negative relationship was found between the area of cystic cavity of the spinal cord and the functional and electrophysiological impairment.
Assuntos
Corantes Fluorescentes/toxicidade , Rosa Bengala/toxicidade , Traumatismos da Medula Espinal/fisiopatologia , Potenciais de Ação/fisiologia , Animais , Eletrofisiologia , Potencial Evocado Motor/fisiologia , Potenciais Somatossensoriais Evocados/fisiologia , Feminino , Atividade Motora/fisiologia , Dor/fisiopatologia , Fotoquímica , Ratos , Ratos Sprague-Dawley , Nervo Isquiático/fisiologia , Medula Espinal/efeitos dos fármacos , Fatores de Tempo , Índices de Gravidade do TraumaRESUMO
BACKGROUND AND PURPOSE: In this study we assessed the relative extent to which resident microglia and blood-borne macrophages contribute to the population of phagocytes after focal infarction of the rat cortex. METHODS: Focal cerebral infarction was induced in rats by photothrombosis after hematogenous macrophages were depleted by means of liposomes containing dichloromethylene diphosphonate. The phagocytic activation of microglia and macrophages was monitored by immunocytochemistry with the antibody ED1. RESULTS: In both macrophage-depleted rats and controls, ED1+ phagocytes bordered the infarct to the same extent at day 3 after photothrombosis. By contrast, at day 6 after photothrombosis ED1+ phagocytes in control rats greatly outnumbered those in macrophage-depleted rats. With the use of the antibody Ox42 directed against the CR3 receptor on the surface of microglia, it was possible to selectively document the transition of resident microglia into stellate and ameboid phagocytic microglia during the first 6 days after photothrombosis in the absence of bloodborne macrophages. CONCLUSIONS: The initial phagocytic response after focal brain ischemia is an intrinsic property of the nervous system mainly performed by resident microglia. The majority of hematogenous macrophages are recruited secondarily to participate in the removal of necrotic tissue.
Assuntos
Infarto Cerebral/patologia , Macrófagos/fisiologia , Microglia/fisiologia , Fagocitose , Animais , Infarto Cerebral/etiologia , Ácido Clodrônico/administração & dosagem , Portadores de Fármacos , Embolia e Trombose Intracraniana/induzido quimicamente , Embolia e Trombose Intracraniana/complicações , Lipossomos , Macrófagos/efeitos dos fármacos , Masculino , Necrose , Fotoquímica , Ratos , Ratos Wistar , Rosa Bengala/efeitos da radiação , Rosa Bengala/toxicidadeRESUMO
The photochemotherapeutic value of topical 8-methoxypsoralen (8-MOP) plus UVA irradiation has been well recognized. The phototoxicity associated with psoralen plus UVA (PUVA) therapy is hallmarked by an increase in vascular permeability (iVP), the accumulation of polymorphonuclear leukocytes (aPMN) and erythema formation in situ. Rose bengal (RB) plus UVA-VIS light (320-700 nm) produces a similar acute inflammatory response, but without immediate or delayed erythema and perceptible edema. This study describes some of the parameters involved in inflammatory reactions evoked by PUVA and the results are compared with RB-induced phototoxic reactions. The rates of iVP and aPMN with a 3 h pulse were quantified using 125I-albumin and 51Cr-labelled PMNs respectively. The erythemal response was graded visually. 8-MOP cream was applied topically, while RB was injected intradermally in rabbit skin before UVA-VIS (9.4 J cm-2) irradiation. The data show that there is no significant difference in the rates of iVP, aPMN and erythema formation between normal skin sites and mast cell-depleted skin sites when challenged with 8-MOP plus light. These results suggest that in situ mast cells do not play a significant role in 8-MOP-photoinduced acute cutaneous inflammatory reactions, in contrast with RB-photoinduced reactions. The iVP and aPMN responses are minimal or absent in sites subjected to repeated exposure to 8-MOP plus light for three or more consecutive days, suggesting the establishment of a desensitized/unresponsive state. Moreover, 8-MOP-photo-desensitized sites do not produce iVP and aPMN of the same magnitude as the normal (naive) skin sites when challenged with RB plus light. Similarly, RB-photo-desensitized sites do not produce iVP and aPMN of the same magnitude as the native skin sites when challenged with 8-MOP plus light. The desensitization and cross-desensitization of skin sites to 8-MOP- or RB-photoinduced reactions suggest that there is either direct attack on the target cell(s), thereby removing the ability to express adhesion molecules, such as endothelial leukocyte adhesion molecule 1 (ELAM-1) or intercellular adhesion molecule 1 (ICAM-1), involved in the accumulation of inflammatory cells, or downregulation of the secretion/release of putative agent(s), such as interleukin 1 (IL-1) and tumor necrosis factor alpha (TNF-alpha), responsible for the initiation and progression of cutaneous inflammations.
Assuntos
Dermatite Fototóxica/imunologia , Ficusina/toxicidade , Rosa Bengala/toxicidade , Animais , Permeabilidade Capilar/efeitos dos fármacos , Permeabilidade Capilar/efeitos da radiação , Dessensibilização Imunológica , Eritema/etiologia , Masculino , Mastócitos/efeitos dos fármacos , Metoxaleno/toxicidade , Neutrófilos/efeitos dos fármacos , Neutrófilos/efeitos da radiação , Terapia PUVA/efeitos adversos , Coelhos , p-Metoxi-N-metilfenetilamina/farmacologiaRESUMO
Reocclusion following thrombolysis is a major limitation of thrombolytic therapy with recombinant tissue-type plasminogen activator (rt-PA) because denuded vessel wall exposed to blood following thrombolysis is a favourable surface for platelet and leucocyte deposition. We have applied a chemiluminescence technique to detect superoxide radical (0(-2)) produced by leucocytes adherent to the femoral artery 24 h after photochemically induced thrombogenesis in the guinea pig in vivo and subsequent thrombolysis by rt-PA. Intravenous administration of MCLA, a specific chemiluminescence reagent for detecting O(-2), markedly increased photon emission. the photon emission was markedly potentiated by phorbol myristate acetate and was suppressed by superoxide dismutase. Reocclusion 24 h after rt-PA induced thrombolysis was observed in 10 of 16 animals. Histological observations revealed extensive polymorphonuclear leucocytes adherent to the vessel wall at the site of thrombogenesis and thrombolysis. A higher level of 0(-2) could be detected from the arteries in which thrombolysis was induced compared with those without thrombolysis. Further, the level 0(-2) detected was greater in reoccluded arteries compared with those in which reflow was established. These observations suggest that 0(-2) is produced by adherent leucocytes at the site of thrombolysis and that leucocytes are involved in reocclusion after thrombolysis.
Assuntos
Endotélio Vascular/metabolismo , Artéria Femoral/metabolismo , Neutrófilos/metabolismo , Ativadores de Plasminogênio/uso terapêutico , Superóxidos/metabolismo , Terapia Trombolítica , Trombose/metabolismo , Animais , Adesão Celular , Modelos Animais de Doenças , Artéria Femoral/patologia , Cobaias , Imidazóis , Luz , Medições Luminescentes , Masculino , Neutrófilos/patologia , Pirazinas , Proteínas Recombinantes/uso terapêutico , Rosa Bengala/toxicidade , Trombose/induzido quimicamente , Trombose/tratamento farmacológico , Trombose/patologiaRESUMO
A large number of experimental studies suggests that oxygen free radicals play a major role in the pathogenesis of the myocardial lesions observed during the sequence ischemia-reperfusion. The purpose of this study was to determine whether oxygen free radicals can induce thrombosis. In so doing we have developed a new experimental thrombosis model. Reproducible focal thrombosis has been achieved by irradiating mesenteric arterioles of rat for variable time with green filtered light issuing from a mercury lamp after systemic injection of different rose bengal doses. The number of emboli that remove in the blood (N), the duration of total occlusion (T) and the number of emboli per minute were then measured. As control, no rose bengal administration was done and the vessels were exposed to the filtered light. In comparison with this control, results clearly showed that free radicals always induced thrombosis and the induced thrombus was mainly composed of platelets. In this new thrombosis model induced by free radicals antithrombotic drugs (aspirin, 200 mg/Kg, heparin, 2 mg/Kg) and antioxidants (vitamin C, 10 and 20 mg/Kg, allopurinol, 200 and 300 mg/Kg, vitamin E, 500 and 1000 mg/Kg) have been tested. Results have shown that only heparin and vitamin E had an antithrombotic effect on thrombus formation induced by free radicals. This model should be useful in studying the effects of different drugs and could lead to new treatment modalities for ischemic accident and other cardiovascular diseases.
Assuntos
Aspirina/uso terapêutico , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Fibrinolíticos/uso terapêutico , Oclusão Vascular Mesentérica/induzido quimicamente , Oxigênio/toxicidade , Rosa Bengala/toxicidade , Alopurinol/farmacologia , Alopurinol/uso terapêutico , Animais , Antioxidantes/farmacologia , Antioxidantes/uso terapêutico , Arteríolas , Ácido Ascórbico/farmacologia , Ácido Ascórbico/uso terapêutico , Coagulação Sanguínea/efeitos dos fármacos , Plaquetas/efeitos dos fármacos , Relação Dose-Resposta a Droga , Fibrinolíticos/farmacologia , Radicais Livres , Heparina/farmacologia , Heparina/uso terapêutico , Masculino , Oclusão Vascular Mesentérica/tratamento farmacológico , Oclusão Vascular Mesentérica/prevenção & controle , Microscopia/instrumentação , Fotoquímica , Ratos , Ratos Wistar , Rosa Bengala/administração & dosagem , Rosa Bengala/efeitos da radiação , Oxigênio Singlete , Gravação de Videoteipe/instrumentação , Vitamina E/farmacologia , Vitamina E/uso terapêuticoRESUMO
We investigated the effect of dietary docosahexaenoic acid (DHA) supplementation on the thrombolytic efficacy of recombinant tissue-type plasminogen activator (rt-PA), platelet aggregability, serum cholesterol and phospholipids. Male Wistar rats (6 weeks old) received dietary DHA supplementation (300 mg/kg per day) for 8 weeks. The rat middle cerebral artery (MCA) was occluded by a thrombus induced by photochemical reaction between rose bengal and green light which cause endothelial damage followed by platelet adhesion, aggregation and formation of a platelet and fibrin-rich thrombus at the site of photochemical reaction. The MCA blood flow was monitored using a laser Doppler flowmeter. rt-PA was administered 30 min after the middle cerebral artery had been occluded by a thrombus. This regimen produced a significant (P < 0.05) decrease in serum free-cholesterol and phospholipids levels, inhibited platelet aggregation ex-vivo induced by collagen in whole blood (P < 0.05), reduced thromboxane (TX) B2 formation (P < 0.01) in whole blood and prolonged the time for thrombotic MCA occlusion (P < 0.01) as compared with values obtained from animals on standard diet. Further, dietary DHA enhanced thrombolytic efficacy of rt-PA and reduced the size of ischaemic cerebral lesions. Our findings suggest that dietary DHA produces antithrombotic effects via metabolic conversion to non-atherogenic and non-platelet stimulant metabolites.