RESUMO
Neurotrauma is among main causes of human disability and death. We studied effects of axotomy on ultrastructure and neuronal activity of a simple model object - an isolated crayfish stretch receptor that consists of single mechanoreceptor neurons (MRN) enwrapped by multilayer glial envelope. After isolation, MRN regularly fired until spontaneous activity cessation. Axotomy did not change significantly MRN spike amplitude and firing rate. However, the duration of neuron activity from MRN isolation to its spontaneous cessation decreased in axotomized MRN relative to intact neuron. [Ca2+] in MRN axon and soma increased 3-10 min after axotomy. Ca2+ entry through ion channels in the axolemma accelerated axotomy-stimulated firing cessation. MRN incubation with Ca2+ionophore ionomycin accelerated MRN inactivation, whereas Ca2+-channel blocker Cd2+ prolonged firing. Activity duration of either intact, or axotomized MRN did not change in the presence of ryanodine or dantrolene, inhibitors of ryanodin-sensitive Ca2+ channels in endoplasmic reticulum. Thapsigargin, inhibitor of endoplasmic reticulum Ca2+-ATPase, or its activator ochratoxin were ineffective. Ultrastructural study showed that the defect in the axon transected by thin scissors is sealed by fused axolemma, glial and collagen layers. Only the 30-50 µm long segment completely lost microtubules and contained swelled mitochondria. The microtubular bundle remained undamaged at 300 µm away from the axotomy site. However, mitochondria within the 200-300 µm segment were strongly condensed and lost matrix and cristae. Glial and collagen layers exhibited greater damage. Swelling and edema of glial layers, collagen disorganization and rupture occurred within this segment. Thus, axotomy stronger damages glia/collagen envelope, axonal microtubules and mitochondria.
Assuntos
Axônios/ultraestrutura , Axotomia , Retículo Endoplasmático/ultraestrutura , Mecanorreceptores/ultraestrutura , Mitocôndrias/ultraestrutura , Animais , Axotomia/métodos , Mecanorreceptores/metabolismo , Neuroglia/citologia , Neurônios/metabolismoRESUMO
Severe mechanical nerve injury such as axotomy can lead to neuron degeneration and death of surrounding glial cells. We showed that axotomy not only mechanically injures glial cells at the cutting location, but also induces necrosis or apoptosis of satellite glial cells remote from the transection site. Therefore, axon integrity is necessary for survival of surrounding glial cells. We used the crayfish stretch receptor that consists of a single mechanoreceptor neuron enveloped by satellite glial cells as a simple, but informative model object in the study of the role of various signaling proteins in axotomy-induced death of remote glial cells. After axon transection, stretch receptors were isolated and incubated in saline in the presence or without specific inhibitors of various signaling proteins. Inhibition of MEK1/2, p38, Akt, GSK-3ß and mTOR increased axotomy-induced apoptosis of remote glial cells, whereas inhibition of ERK1/2 and GSK-3ß enhanced necrosis. This suggests the involvement of these signaling proteins in protective, antiapoptotic and antinecrotic processes in the remote satellite glia surrounding the axotomized mechanoreceptor neuron.
Assuntos
Apoptose , Axônios/metabolismo , Sistema de Sinalização das MAP Quinases , Mecanorreceptores/metabolismo , Neuroglia/metabolismo , Quinases Proteína-Quinases Ativadas por AMP , Animais , Astacoidea , Células Cultivadas , Glicogênio Sintase Quinase 3 beta/metabolismo , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 2/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Necrose , Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
BACKGROUND: Photodynamic therapy (PDT) is used for cancer treatment including brain tumors. But the role of epigenetic processes in photodynamic injury of normal brain tissue is unknown. METHODS: 5-Aminolevulinic acid (ALA), a precursor of protoporphyrin IX (PpIX), was used to photosensitize mouse cerebral cortex. PpIX accumulation in cortical tissue was measured spectrofluorometrically. Hematoxylin/eosin, gallocyanin-chromalum and immunohistochemical staining were used to study morphological changes in PDT-treated cerebral cortex. Proteomic antibody microarrays were used to evaluate expression of 112 proteins involved in epigenetic regulation. RESULTS: ALA administration induced 2.5-fold increase in the PpIX accumulation in the mouse brain cortex compared to untreated mice. Histological study demonstrated PDT-induced injury of some neurons and cortical vessels. ALA-PDT induced dimethylation of histone H3, upregulation of histone deacetylases HDAC-1 and HDAC-11, and DNA methylation-dependent protein Kaiso that suppressed transcriptional activity. Upregulation of HDAC-1 and H3K9me2 was confirmed immunohistochemically. Down-regulation of transcription factor FOXC2, PABP, and hBrm/hsnf2a negatively regulated transcription. Overexpression of phosphorylated histone H2AX indicated activation of DNA repair, but down-regulation of MTA1/MTA1L1 and PML - impairment of DNA repair. Overexpression of arginine methyltransferase PRMT5 correlated with up-regulation of transcription factor E2F4 and importin α5/7. CONCLUSION: ALA-PDT injures and kills some but not all neurons and caused limited microvascular alterations in the mouse cerebral cortex. It alters expression of some proteins involved in epigenetic regulation of transcription, histone modification, DNA repair, nuclear protein import, and proliferation. GENERAL SIGNIFICANCE: These data indicate epigenetic markers of photo-oxidative injury of normal brain tissue.
Assuntos
Ácido Aminolevulínico/farmacologia , Córtex Cerebral/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Regulação da Expressão Gênica , Fotoquimioterapia , Fármacos Fotossensibilizantes/farmacologia , Proteoma/análise , Animais , Córtex Cerebral/patologia , Córtex Cerebral/efeitos da radiação , Epigênese Genética/genética , Epigênese Genética/efeitos da radiação , Epigenômica , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos da radiação , Histonas/metabolismo , Técnicas Imunoenzimáticas , Masculino , Camundongos , Análise Serial de ProteínasRESUMO
Photodynamic impact on animal cerebral cortex using water-soluble Bengal Rose as a photosensitizer, which does not cross the blood-brain barrier and remains in blood vessels, induces platelet aggregation, vessel occlusion, and brain tissue infarction. This reproduces ischemic stroke. Irreversible cell damage within the infarction core propagates to adjacent tissue and forms a transition zone - the penumbra. Tissue necrosis in the infarction core is too fast (minutes) to be prevented, but much slower penumbral injury (hours) can be limited. We studied the changes in morphology and protein expression profile in penumbra 1 h after local photothrombotic infarction induced by laser irradiation of the cerebral cortex after Bengal Rose administration. Morphological study using standard hematoxylin/eosin staining showed a 3-mm infarct core surrounded by 1.5-2.0 mm penumbra. Morphological changes in the penumbra were lesser and decreased towards its periphery. Antibody microarrays against 224 neuronal and signaling proteins were used for proteomic study. The observed upregulation of penumbra proteins involved in maintaining neurite integrity and guidance (NAV3, MAP1, CRMP2, PMP22); intercellular interactions (N-cadherin); synaptic transmission (glutamate decarboxylase, tryptophan hydroxylase, Munc-18-1, Munc-18-3, and synphilin-1); mitochondria quality control and mitophagy (PINK1 and Parkin); ubiquitin-mediated proteolysis and tissue clearance (UCHL1, PINK1, Parkin, synphilin-1); and signaling proteins (PKBα and ERK5) could be associated with tissue recovery. Downregulation of PKC, PKCß1/2, and TDP-43 could also reduce tissue injury. These changes in expression of some neuronal proteins were directed mainly to protection and tissue recovery in the penumbra. Some upregulated proteins might serve as markers of protection processes in a penumbra.
Assuntos
Isquemia Encefálica/metabolismo , Trombose Intracraniana/metabolismo , Proteômica/métodos , Animais , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Caderinas/metabolismo , Córtex Cerebral/irrigação sanguínea , Córtex Cerebral/metabolismo , Infarto Cerebral/metabolismo , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Masculino , Ratos , Transdução de SinaisRESUMO
Photodynamic therapy (PDT) is used for cancer treatment, including brain tumors. To explore the dynamics of photodynamic injury of glial cells and neurons and corresponding neuroglial interactions, we studied ultrastructure of the PDT-treated crayfish stretch receptor that consists of a single sensory neuron enwrapped by glial cells. Just after PDT, swelling of some mitochondria, dictyosomes, and endoplasmic reticulum cisterns occurred in neurons and glial cells. Tubular lattices involved in intraglial transport became swollen and disintegrated. At 1 hr postirradiation, these alterations were expanded to the whole cells. Segregation of the neuronal cytoplasm by Nissl bodies, which were involved in protein synthesis and transport along neurites, was lost. Swelling of submembrane cisterns prevented formation of glial protrusions and double-wall vesicles involved in the glia-to-neuron transport. Five hours later, glial layers lost organelles, stuck together, or dilated locally as a result of edema. In the neuronal cytoplasm, only demises of ER and swollen mitochondria were present, but few mitochondria retained normal structure. Thus, swelling of intracellular organelles, the first sign of photodynamic injury, occurred simultaneously in neurons and glia, but glial organelles were eliminated more quickly. Therefore, glial cells were less resistant to PDT than neurons. Adjacent glial layers were damaged less than remote ones, suggesting their protection by the neuron. The structures involved in glia-to-neuron (neuronal submembrane cisterns, glial protrusions, double-wall vesicles), intraglial (tubular lattices), and intraneuronal (Nissl bodies, Golgi apparatus, microtubular bundles) transport were impaired at the earlier stages of stretch receptor damage.
Assuntos
Astacoidea/anatomia & histologia , Neuroglia/fisiologia , Neuroglia/ultraestrutura , Neurônios/fisiologia , Neurônios/ultraestrutura , Animais , Luz , Microscopia Eletrônica de Transmissão/métodos , Músculos/citologia , Neuroglia/efeitos da radiação , Neurônios/efeitos da radiação , Transporte Proteico/fisiologia , Transporte Proteico/efeitos da radiaçãoRESUMO
Unilateral photothrombotic stroke caused tissue infarct in the mouse cerebral cortex. The injury of the cerebral cortex impaired the mouse motor activity, in particular the functional asymmetry in forelimb use. In the peri-infarct cortical tissue outside the infarct core cell apoptosis occurred at 4 and 7 days after PTS. The downregulation of acetylated α-tubulin, a marker of stable microtubules, showed the destruction of neurites, axons, and dendrites in injured neurons. However, the upregulation of GAP43 indicates the stimulation of neurite growth that was possibly aimed at the recovery of the cortical tissue in the damaged cerebral hemisphere. Application of MI-192, an inhibitor of histone deacetylases HDAC2 and HDAC3, demonstrated the neuroprotective activity in the mouse brain subjected to photothrombotic stroke. It reduced the volume of the PTS-induced infarction core in the mouse brain, partly restored the functional symmetry in the forelimb use, decreased the level of PTS-induced apoptosis and acetylation of α-tubulin characteristic for stable microtubules, and increased the expression of GAP-43 in the cerebral cortex of the damaged hemisphere. These data point to the involvement of HDAC2 and HDAC3 in the photothrombotic injury of the mouse brain not only in the infarction core but also outside it. The application of MI192 after PTS reduced or eliminated these negative effects and exerted the neuroprotective effect on the mouse brain. It may be a promising neuroprotector agent for anti-stroke therapy.
Assuntos
Benzamidas/uso terapêutico , Encéfalo/patologia , Inibidores de Histona Desacetilases/uso terapêutico , Luz , Fármacos Neuroprotetores/uso terapêutico , Acidente Vascular Cerebral/tratamento farmacológico , Trombose/tratamento farmacológico , Acetilação/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Comportamento Animal/efeitos dos fármacos , Benzamidas/química , Benzamidas/farmacologia , Encéfalo/efeitos dos fármacos , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/patologia , Proteína GAP-43/metabolismo , Histona Desacetilase 2/antagonistas & inibidores , Histona Desacetilase 2/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Histonas/metabolismo , Masculino , Camundongos , Fármacos Neuroprotetores/farmacologia , Acidente Vascular Cerebral/complicações , Trombose/complicações , Tubulina (Proteína)/metabolismoRESUMO
Stroke is one of the leading reasons of human death. Ischemic penumbra that surrounds the stroke-induced infarction core is potentially salvageable, but molecular mechanisms of its formation are poorly known. Histone acetylation induces chromatin decondensation and stimulates gene expression. We studied the changes in the levels and localization of histone acetyltransferases HAT1 and PCAF in penumbra after photothrombotic stroke (PTS, a stroke model). In PTS, laser irradiation induces local occlusion of cerebral vessels after photosensitization by Rose Bengal. HAT1 and PCAF are poorly expressed in normal cortical neurons and astrocytes, but they are overexpressed 4-24 h after PTS. Their predominant localization in neuronal nuclei did not change after PTS, but their levels in the astrocyte nuclei significantly increased. Western blotting showed the increase of HAT1 and PCAF levels in the cytoplasmic fraction of the PTS-induced penumbra. In the nuclear fraction, PCAF level did not change, and HAT1 was overexpressed only at 24 h post-PTS. PTS-induced upregulation of HAT1 and PCAF in the penumbra was mainly associated with overexpression in the cytoplasm of neurons and especially astrocytes. HAT1 and PCAF did not co-localize with TUNEL-positive cells that indicated their nonparticipation in PTS-induced apoptosis.
Assuntos
Astrócitos/metabolismo , Córtex Cerebral/metabolismo , Histona Acetiltransferases/metabolismo , Neurônios/metabolismo , AVC Trombótico/metabolismo , Fatores de Transcrição de p300-CBP/metabolismo , Animais , Astrócitos/patologia , Núcleo Celular/metabolismo , Córtex Cerebral/patologia , Citoplasma/metabolismo , Masculino , Neurônios/patologia , Ratos , Ratos Wistar , AVC Trombótico/patologiaRESUMO
Epigenetic processes play important roles in brain responses to ischemic injury. We studied effects of photothrombotic stroke (PTS, a model of ischemic stroke) on the intracellular level and cellular localization of histone deacetylases HDAC3, HDAC4 and HDAC6 in the rat brain cortex, and tested the potential neuroprotector ability of their inhibitors. The background level of HDAC3, HDAC4 and HDAC6 in the rat cerebral cortex was relatively low. HDAC3 localized in the nuclei of some neurons and few astrocytes. HDAC4 was found in the neuronal cytoplasm. After PTS, their levels in penumbra did not change, but HDAC4 appeared in the nuclei of some cells. Its level in the cytoplasmic, but not nuclear fraction of penumbra decreased at 24, but not 4 h after PTS. HDAC6 was upregulated in neurons and astrocytes in the PTS-induced penumbra, especially in the nuclear fraction. Unlike HDAC3 and HDAC4, HDAC6 co-localized with TUNEL-positive apoptotic cells. Inhibitory analysis confirmed the involvement of HDAC6, but not HDAC3 and HDAC4 in neurodegeneration. HDAC6 inhibitor HPOB, HDAC2/8 inhibitor α-phenyl tropolone, and non-specific histone deacetylase inhibitor sodium valproate, but not HDAC3 inhibitor BRD3308, or HDAC4 inhibitor LMK235, decreased PTS-induced infarction volume in the mouse brain, reduced apoptosis, and recovered the motor behavior. HPOB also restored PTS-impaired acetylation of α-tubulin. α-phenyl tropolone restored acetylation of histone H4 in penumbra cells. These results suggest that histone deacetylases HDAC6 and HDAC2 are the possible molecular targets for anti-ischemic therapy, and their inhibitors α-phenyl tropolone, HBOP and sodium valproate can be considered as promising neuroprotectors.
Assuntos
Córtex Cerebral/enzimologia , Desacetilase 6 de Histona/biossíntese , Inibidores de Histona Desacetilases/farmacologia , Trombose Intracraniana/enzimologia , Acidente Vascular Cerebral/enzimologia , Tropolona/farmacologia , Ácido Valproico/farmacologia , Animais , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/patologia , Expressão Gênica , Inibidores de Histona Desacetilases/uso terapêutico , Histona Desacetilases/biossíntese , Trombose Intracraniana/patologia , Trombose Intracraniana/prevenção & controle , Lasers Semicondutores/efeitos adversos , Masculino , Camundongos , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Ratos , Acidente Vascular Cerebral/patologia , Acidente Vascular Cerebral/prevenção & controle , Tropolona/uso terapêutico , Ácido Valproico/uso terapêuticoRESUMO
In ischemic stroke, vascular occlusion rapidly induces tissue infarct. Over the ensuing hours, damage spreads to adjacent tissue and forms transition zone (penumbra), which is potentially salvageable. Epigenetic regulation of chromatin structure controls gene expression and protein synthesis. We studied the expression of histone deacetylases HDAC1 and HDAC2 in the penumbra at 4 or 24 h after photothrombotic stroke (PTS) in the rat brain cortex. PTS increased the expression of HDAC1 and HDAC2 in penumbra and caused the redistribution of HDAC1 but not HDAC2 from the neuronal nuclei to cytoplasm. In astrocytes, HDAC1 expression and localization did not change. In neurons, HDAC2 localized exclusively in nuclei, but in astrocytes, it was also observed in processes. PTS induced neuronal apoptosis in the penumbra. TUNEL-stained apoptotic neurons co-localized with HDAC2 but not HDAC1. These data suggest that HDAC2 may represent the potential target for anti-stroke therapy and its selective inhibition may be a promising strategy for the protection of the penumbra tissue after ischemic stroke.
Assuntos
Apoptose , Histona Desacetilase 1/metabolismo , Histona Desacetilase 2/metabolismo , Luz , Acidente Vascular Cerebral/complicações , Acidente Vascular Cerebral/enzimologia , Trombose/complicações , Trombose/enzimologia , Animais , Astrócitos/metabolismo , Biomarcadores/metabolismo , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Proteína Glial Fibrilar Ácida/metabolismo , Masculino , Ratos WistarRESUMO
Photodynamic therapy (PDT) is used for treatment of cancer, including brain tumors. To explore the mechanism of photodynamic injury of neurons, we studied the PDT effect of 10(-7) M Photosens on ultrastructure of isolated crayfish stretch receptor neuron that was used as a model object. After a 5-min treatment that only slightly changed neuron activity, the initial injury (alteration of some mitochondria, vacuolization of the cytoplasm) was observed in parallel with compensatory changes (chromatin decondensation, elongation and aggregation of mitochondria, formation of lysosomes and autophagosomes). Longer photosensitization (30 min) abolished firing, destroyed mitochondria and Golgi apparatus, depleted energy sources (glycogen granules), and impaired granular endoplasmic reticulum and polysomes involved in protein synthesis. Therefore, mitochondria and Golgi apparatus were the primary targets for Photosens-mediated PDT in a single neuron. Their alteration might underlie functional shifts. These structural changes continued to develop after abolishment of neuronal activity and led to necrosis.
Assuntos
Indóis/farmacologia , Mecanorreceptores/efeitos dos fármacos , Mecanorreceptores/ultraestrutura , Neurônios/efeitos dos fármacos , Neurônios/ultraestrutura , Compostos Organometálicos/farmacologia , Radiossensibilizantes/farmacologia , Animais , Astacoidea , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/ultraestrutura , Microscopia Eletrônica de Transmissão , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/ultraestrutura , FotoquimioterapiaRESUMO
Chlorin e(6) and its derivatives are promising sensitizers for photodynamic therapy (PDT). In order to compare the photodynamic effects of 8 novel derivatives of chlorin e(6) and to explore some mechanisms of their effects at the cellular level, we studied PDT-induced changes in bioelectric activity of crayfish mechanoreceptor neuron that was used as a sensitive experimental model. Neurons were insensitive to red laser irradiation (632.8 nm; 0.3 W/cm(2)) or to photosensitizers alone, but changed firing rate and died under the photodynamic effect of nanomolar concentrations of sensitizers. The dynamics of neuron responses depended on photosensitizer type and concentration. The dependence of neuron lifetime on photosensitizer concentration allowed comparing efficiencies of different photosensitizers. Radachlorin was the most potent photosensitizer comparable with mTHPC. High photodynamic efficiency of some chlorin e(6) derivatives was related to weak dependence of neuron lifetime on sensitizer concentration, indicating to the initiation of 2-3 secondary processes such as free radical membrane damage by one absorbed photon. Photodynamic efficiency of sensitizers depended on amphiphilicity influencing their intracellular localization.
Assuntos
Astacoidea/fisiologia , Mecanorreceptores/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Fármacos Fotossensibilizantes/farmacologia , Porfirinas/farmacologia , Animais , Morte Celular/efeitos dos fármacos , Clorofilídeos , Relação Dose-Resposta a Droga , Eletrofisiologia , Técnicas In Vitro , Lasers , Fármacos Fotossensibilizantes/química , Porfirinas/química , Relação Estrutura-AtividadeRESUMO
The photodynamic effects of sulphonated zinc and aluminum phthalocyanine derivatives as well as phosphonated aluminum phthalOcyanine on the firing of isolated crayfish mechanoreceptor neurons were studied. After 30 min staining neurons were irradiated with He-Ne laser (632.8 nm, 0.3 W/cm2) and changes in neuron firing frequency were recorded. Neuron firing was found to be very sensitive to photodynamic effect and could serve as a sensitive indicator of cell photodamage. It changed the firing level and then died at nanomolar concentrations of phthalocyanines. The dynamics of the neuron responses to photodynamic effects included stages of firing activation and/or inhibition prior to irreversible firing abolition. The order of these stages depended on photosensitizer type and concentration. The comparison of the dependencies of neuron lifetime on photosensitizer concentrations showed ZnPcS2 to be the most effective photosensitizer.
Assuntos
Indóis/farmacologia , Mecanorreceptores/fisiologia , Neurônios/fisiologia , Compostos Organometálicos/farmacologia , Fármacos Fotossensibilizantes/farmacologia , Alumínio/farmacologia , Animais , Astacoidea , Técnicas In Vitro , Isoindóis , Lasers , Neurônios/efeitos dos fármacos , Neurônios/efeitos da radiação , Relação Estrutura-Atividade , Zinco/farmacologia , Compostos de ZincoRESUMO
Epigenetic processes are involved in regulation of cell functions and survival, but their role in responses of neurons and glial cells to oxidative injury is insufficiently explored. Here, we studied the role of DNA methylation and histone deacetylation in reactions of neurons and surrounding glial cells to photodynamic treatment that induces oxidative stress and cell death. Isolated crayfish stretch receptor consisting of a single mechanoreceptor neuron surrounded by glial cells was photosensitized with aluminum phthalocyanine Photosens that induced neuron inactivation, necrosis of the neuron and glia, and glial apoptosis. Inhibitors of DNA methylation 5-azacytidine and 5-aza-2'-deoxycytidine (decitabine) reduced the level of PDT-induced necrosis of glial cells but not neurons by 1.3 and 2.0 times, respectively, and did not significantly influence apoptosis of glial cells. Histone deacetylase inhibitors valproic acid and trichostatin A inhibited PDT-induced both necrosis and apoptosis of satellite glial cells but not neurons by 1.6-2.7 times. Thus, in the crayfish stretch receptor DNA methylation and histone deacetylation are involved in epigenetic control of glial but not neuronal necrosis. Histone deacetylation also participates in glial apoptosis.
Assuntos
Apoptose/genética , Mecanorreceptores/efeitos dos fármacos , Mecanorreceptores/efeitos da radiação , Necrose/genética , Neuroglia/efeitos dos fármacos , Neuroglia/efeitos da radiação , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/efeitos da radiação , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Apoptose/efeitos da radiação , Astacoidea , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Metilação de DNA/efeitos dos fármacos , Metilação de DNA/efeitos da radiação , Decitabina , Inibidores Enzimáticos/farmacologia , Epigênese Genética/efeitos dos fármacos , Epigênese Genética/efeitos da radiação , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Técnicas In Vitro , Indóis/farmacologia , Lasers , Mecanorreceptores/fisiologia , Necrose/induzido quimicamente , Necrose/tratamento farmacológico , Necrose/fisiopatologia , Neuroglia/fisiologia , Compostos Organometálicos/farmacologia , Estimulação Luminosa/efeitos adversos , Fármacos Fotossensibilizantes/farmacologia , Ácido Valproico/farmacologiaRESUMO
BACKGROUND: Radachlorin, a chlorine-derived photosensitizer, is used currently in photodynamic therapy (PDT) of skin cancer. In this work we studied Radachlorin-PDT effect on peripheral nerve and glial cells that are damaged along with tumor tissue. METHODS: We used simple model objects - a crayfish stretch receptor that consists of a single sensory neuron surrounded by glial cells and crayfish nerve cord consisting of nerve fibers and ganglia. Radachlorin absorption and emission spectra were registered using spectrophotometer and spectrofluorimeter. Radachlorin accumulation and intracellular localization were studied using the fluorescence microscope. Necrotic and apoptotic cells were visualized using propidium iodide and Hoechst 33342. Neuronal activity was registered using standard electrophysiological methods. RESULTS: Radachlorin absorption spectrum in the physiological van Harreveld saline (pH 7.3) contained maximums at 420 and 654nm. Its fluorescence band 620-700nm had a maximum at 664nm. In the crayfish stretch receptor Radachlorin localized predominantly to the glial envelope and penetrated slightly into the neuron body and axon. Radachlorin rapidly accumulated in the crayfish nerve cord tissue within 30min. Its elimination in the dye-free solution occurred slower: 11% loss for 2h. Radachlorin-PDT inactivated the neuron and induced necrosis of neurons and glial cells and glial apoptosis at concentrations as low as 10(-10)-10(-9)M. CONCLUSIONS: Radachlorin rapidly accumulates in the nervous tissue, mainly in glial cells, and demonstrates very high photodynamic efficacy that characterize it as a promising photosensitizer.
Assuntos
Neuroglia/fisiologia , Neurônios/fisiologia , Fotoquimioterapia/métodos , Porfirinas/administração & dosagem , Porfirinas/farmacocinética , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Astacoidea , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Relação Dose-Resposta a Droga , Combinação de Medicamentos , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Neuroglia/citologia , Neuroglia/efeitos dos fármacos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Fármacos Fotossensibilizantes/administração & dosagem , Fármacos Fotossensibilizantes/farmacocinéticaRESUMO
Photodynamic treatment that causes intense oxidative stress and cell death is currently used in neurooncology. However, along with tumor cells, it may damage healthy neurons and glia. In order to study photodynamic effect on normal nerve and glial cells, we used crayfish stretch receptor, a simple system consisting of only two identified sensory neurons surrounded by glial cells. Photodynamic treatment induced firing abolition and necrosis of neurons as well as necrosis and apoptosis of glial cells. Nerve growth factor but not brain-derived neurotrophic factor or epidermal growth factor protected glial cells but not neurons from photoinduced necrosis and apoptosis. Inhibitors of tyrosine kinases or protein kinase JNK eliminated anti-apoptotic effect of nerve growth factor in photosensitized glial cells but not neurons. Therefore, these signaling proteins were involved in the anti-apoptotic activity of nerve growth factor. These data indicate the possible presence of receptors capable of recognizing murine nerve growth factor in crayfish glial cells. Thus, intercellular signaling mediated by nerve-growth-factor-like neurotrophin, receptor tyrosine kinase, and JNK may be involved in crayfish glia protection from apoptosis induced by photodynamic treatment.
Assuntos
Astacoidea , Luz/efeitos adversos , Mecanorreceptores/efeitos da radiação , Fatores de Crescimento Neural/metabolismo , Neuroglia/efeitos da radiação , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Morte Celular/fisiologia , Fator de Crescimento Epidérmico/metabolismo , Feminino , Humanos , Indóis/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Masculino , Mecanorreceptores/citologia , Mecanorreceptores/metabolismo , Neuroglia/citologia , Neuroglia/metabolismo , Compostos Organometálicos/metabolismo , Radiossensibilizantes/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
To study the involvement of neuroglial interactions in photodynamic damage of crayfish stretch receptor, which consists of only two neurons surrounded by satellite glial cells (SGCs), we attempted to proteolytically uncouple neurons and glia and then compare the responses of these cells to photosensitization when intercellular communications were intact or impaired. After incubation of isolated stretch receptors with pronase or collagenase they were photosensitized with Photosens, a mixture of sulfonated alumophthalocyanines AlPcS(n) (n = 2, 3, and 4; mean n = 3.1). In the next 6 hr the preparations were double fluorochromed with propidium iodide and Hoechst-33342 to visualize necrotic and apoptotic cells. Proteolytic treatment shortened bioelectric neuron response and precipitated its functional inactivation; however, it did not significantly impair neuron morphology and did not induce its necrosis either in the darkness or under photosensitization. Photodynamic treatment induced necrosis of neurons and SGC and apoptosis of glial cells. Pronase but not collagenase increased percent of necrotic and apoptotic SGCs in the darkness and thus reduced the number of glial cells around the neuron; however, both pronase and collagenase prevented photodynamically induced apoptosis of glial cells. The involvement of neuron-to-glia signaling interactions in this phenomenon is suggested.
Assuntos
Mecanorreceptores , Neuroglia/efeitos dos fármacos , Neurônios Aferentes/efeitos dos fármacos , Peptídeo Hidrolases/farmacologia , Triazenos/farmacologia , Potenciais de Ação/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Astacoidea , Contagem de Células/métodos , Colagenases/farmacologia , Relação Dose-Resposta a Droga , Interações Medicamentosas , Técnicas In Vitro , Neuroglia/citologia , Neurônios Aferentes/citologia , Pronase/farmacologia , Fatores de TempoRESUMO
Effects of photodynamic therapy (PDT) on adhesion of human adenocarcinoma cells of the line WiDr to a plastic substratum were investigated. Protoporphyrin IX induced by 5-aminolevulinic acid (ALA) was used as a photosensitizer. Light exposure inhibited attachment of suspended cells to a substratum. The adhesion was most strongly pronounced for light exposures around 200 mJ/cm(2) causing cell death. However, sub-lethal exposures (42 mJ/cm(2), 97% survival) inhibited cell adhesion as well. Sub-lethal ALA-PDT increased the intracellular space in dense colonies of WiDr cells. This was attributed to formation of lamellipodia between the cells and to increased numbers of focal contacts containing alpha(V)beta(3) integrin in some of the cells. The E-cadherin distribution was not changed by the treatment. Complex processes, including changes in cellular shape and reorganization of the cytoskeleton, are suggested to participate in the observed ALA-PDT effect on the cell adhesion.
Assuntos
Neoplasias/tratamento farmacológico , Fármacos Fotossensibilizantes/farmacologia , Protoporfirinas/farmacologia , Caderinas/metabolismo , Adesão Celular/efeitos dos fármacos , Imunofluorescência , Humanos , Integrina alfaVbeta3/metabolismo , Luz , Fatores de Tempo , Células Tumorais CultivadasRESUMO
The photodynamic effects of 6 new deuteroporphyrin IX derivatives with different amphiphilicity and lipophilicity, as well as effects of known hematoporphyrin derivatives Photofrin II and Photoheme on isolated crayfish mechanoreceptor neurons were studied. After 30 min photosensitization, neurons were irradiated with He-Ne laser (632.8 nm, 0.3 W/cm(2)), and changes in their firing frequency were recorded. Neuron firing was shown to be very sensitive to photodynamic effect of the studied deuteroporphyrin IX derivatives causing irreversible firing abolition at pikomolar concentrations while Photoheme and Photofrin II were effective in the nanomolar range. The most effective sensitizers were 4-(1-methyl-3-hydroxybutyl)- and 4-(1-methyl-2-acetyl-3-oxobutyl)-deuteroporphyrins. Extinction and amphiphilicity were shown to be the most important properties determining photodynamic efficiency of the studied photosensitizers.
Assuntos
Deuteroporfirinas/farmacologia , Hematoporfirinas/farmacologia , Neurônios/efeitos dos fármacos , Fármacos Fotossensibilizantes/farmacologia , Potenciais de Ação/efeitos dos fármacos , Animais , Astacoidea , Morte Celular , Deuteroporfirinas/química , Éter de Diematoporfirina/química , Éter de Diematoporfirina/farmacologia , Relação Dose-Resposta a Droga , Hematoporfirinas/química , Neurônios/fisiologia , Técnicas de Cultura de Órgãos , Fármacos Fotossensibilizantes/químicaRESUMO
Hypericin, a natural polycyclic quinone extracted from Hypericum perforatum, has been recently shown to be a powerful sensitiser for photodynamic therapy (PDT). However, its intracellular localisation remains unclear and contradictory. In the present work we compared the intracellular localisation of hypericin in three cultured cell lines (adenocarcinoma cells WiDr, carcinoma cells NHIK 3025 and glioblastoma cells D54Mg) with the distribution of fluorescent probes specific to lysosomes (LysoTracker Blue DND-22), mitochondria (MitoTracker Green FM) and endoplasmic reticulum (ERTracker Blue-White DPX). It was shown that the hypericin staining pattern was different compared to the intracellular distribution of mitochondria or lysosomes. Hypericin was concentrated in the perinucleolar cytoplasmic area mainly on one side of the nucleus--the region rich in endoplasmic reticulum and Golgi. Sometimes nuclear envelope was also stained. Plasma membrane was not stained but the dye was often accumulated in the intercellular space between the tightly contacting WiDr cells in colonies. Hypericin concentrations of 10 microM or less were not toxic for WiDr cells in the dark. Orange light (lambda max approximately 600 nm; 6 mW/cm2) killed the cells stained with 1 microM hypericin with LD50 approximately 1 J/cm2.