RESUMEN
Anion exchange polystyrene nanofiber materials (AE) were prepared by electrospinning followed by two-step functionalization of the nanofiber surface by chlorosulfonic acid and ethylendiamine. The photoactive character of these materials was introduced through adsorption of the tetra-anionic 5,10,15,20-tetrakis-(4-sulfonatophenyl)porphyrin photosensitizer (TPPS-AE) on the nanofiber surface or by encapsulation of the nonpolar 5,10,15,20-tetraphenylporphyrin photosensitizer (AE(TPP)) into the nanofibers. Anion exchange nanofiber materials with porphyrins are characterized by a high ion-exchange capacity, photogeneration of singlet oxygen O2((1)Δg), and singlet oxygen-sensitized delayed fluorescence. Due to the photogeneration of cytotoxic O2((1)Δg), the nanofibers exhibited oxidation of the external substrates in aqueous solution and an efficient antibacterial effect when activated by simulated daylight. Adsorption of both TPPS and I(-) on the surface of AE led to the formation of more efficient I-TPPS-AE materials. Rapid photooxidation of I(-) by O2((1)Δg), and the formation of another cytotoxic species, I3(-), on the surface of the nanofibers were responsible for the increased antibacterial properties of I-TPPS-AE and the prolonged antibacterial effect in the dark.
Asunto(s)
Luz , Nanofibras/química , Fármacos Fotosensibilizantes/química , Porfirinas/química , Adsorción , Aniones/química , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Escherichia coli/efectos de la radiación , Intercambio Iónico , Cinética , Oxidación-Reducción , Fármacos Fotosensibilizantes/farmacología , Poliestirenos/química , Porfirinas/farmacología , Oxígeno Singlete/química , Oxígeno Singlete/metabolismoRESUMEN
Photodynamic therapy (PDT) is an alternative method of tumour treatment. It is based on a photochemical reaction of a photosensitizer, irradiation, and O(2) which converts to cytotoxic (1)O(2) and other forms of reactive oxygen species (ROS). The comet assay (also called single-cell gel electrophoresis, SCGE) is a sensitive, simple and quantitative technique for detection of DNA damage. In our study we investigated the phototoxicity of the two porphyrin photosensitizers, TPPS4 and MgTPPS4, on HeLa cells. Three different radiation doses and six different concentrations of the photosensitizers were used. Our results show that the DNA of the cells treated with the TPPS(4) and MgTPPS(4) at the concentrations higher than 5 µM was highly fragmented indicating a strong phototoxic effect resulting in a cell apoptosis. On the base of our results we can hypothesize that even the irradiation dose of 1 J cm(-2) is sufficient enough to provoke the DNA fragmentation.
Asunto(s)
Fragmentación del ADN/efectos de los fármacos , Metaloporfirinas/farmacología , Fármacos Fotosensibilizantes/farmacología , Porfirinas/farmacología , Apoptosis/efectos de los fármacos , Ensayo Cometa , Relación Dosis-Respuesta a Droga , Células HeLa , Humanos , Magnesio/química , Metaloporfirinas/administración & dosificación , Metaloporfirinas/química , Fotoquimioterapia/métodos , Fármacos Fotosensibilizantes/administración & dosificación , Porfirinas/administración & dosificación , Porfirinas/química , Dosis de RadiaciónRESUMEN
The objectives of this study was to investigate the production of reactive oxygen species (ROS) after photodynamic therapy (PDT) in vitro. We examined second generation sensitizers, porphyrines (TPPS4, ZnTPPS4 and PdTPPS4) and compared their effectivity on ROS generation in G361 cell line. Used porphyrines are very efficient water-soluble aromatic dyes with potential to use in photomedicine and have a high propensity to accumulate in the membranes of intracellular organelles like lysosomes and mitochondria. Interaction between the triplet excited state of the sensitizer and molecular oxygen leads to produce singlet oxygen and other ROS to induce cell death. Production of ROS was verificated by molecular probe CM-H2DCFDA and viability of cells was determined by MTT assay. Our results demonstrated that ZnTPPS4 induces the highest ROS production in cell line compared to TPPS4 and PdTPPS4 at each used concentration and light dose. These results consist with a fact that photodynamic effect depends on sensitizer type, its concentration and light dose.
Asunto(s)
Paladio , Fotoquimioterapia , Fármacos Fotosensibilizantes/farmacología , Porfirinas/farmacología , Especies Reactivas de Oxígeno/metabolismo , Zinc , Línea Celular Tumoral , Humanos , MelanomaRESUMEN
The production of reactive oxygen species (ROS) has a crucial effect on the result of photodynamic therapy (PDT). Because of this fact, we examined the ROS formation by means of three porphyrin sensitizers (TPPS(4), ZnTPPS(4) and PdTPPS(4)) and compared their effectivity for induction of cell death in the G361 (human melanoma) cell line. The porphyrins used are very efficient water-soluble aromatic dyes with a potential application in photomedicine and have a high tendency to accumulate in the membranes of intracellular organelles such as lysosomes and mitochondria. Interaction between the triplet excited state of the sensitizer and molecular oxygen leads to the production singlet oxygen and other reactive oxygen species to induce cell death. Production of ROS was investigated by molecular probe CM-H(2)DCFDA. Our results demonstrated that ZnTPPS(4) induces the highest ROS production in the cell line compared to TPPS(4) and PdTPPS(4) at concentrations of 1, 10, and 100 microM and light dose of 1 J cm(-2). We also observed a consequence between ROS production and cell survival. In conclusion, these results demonstrate that photodynamic effect depends on sensitizer type, its concentration and light dose.
Asunto(s)
Fotoquimioterapia/métodos , Fármacos Fotosensibilizantes/farmacología , Porfirinas/farmacología , Especies Reactivas de Oxígeno/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Luz , Melanoma/metabolismo , Metaloporfirinas/administración & dosificación , Metaloporfirinas/farmacología , Paladio/administración & dosificación , Paladio/química , Paladio/farmacología , Fármacos Fotosensibilizantes/administración & dosificación , Porfirinas/administración & dosificación , Zinc/farmacologíaRESUMEN
The first part of this article is devoted to a minireview of basic terms of plasma physics and chemistry described in a way understandable even to nonspecialists in the field. Among the methods of generation of low-temperature plasma, the unipolar electric corona discharge in air at atmospheric pressure is described in more detail. Selected studies are mentioned that concern the decontamination effects of low-temperature plasma and the currently known mechanisms of its microbicidal action. The key part of this mechanism is the action of UV light and both charged and uncharged particles. The most important common mechanism seems to be different forms of reactive oxygen species and some methods of their determination are therefore briefly described.
Asunto(s)
Descontaminación , Electricidad , Bacterias/crecimiento & desarrollo , Bacterias/efectos de la radiación , Descontaminación/métodos , Gases , Temperatura , Rayos UltravioletaRESUMEN
The second part of our paper presents the results of experiments with the decontamination of surfaces by low-temperature plasma generated by corona discharge in air at atmospheric pressure. A simple device is described and the effects of the corona discharge on model microorganisms, viz. the yeast Candida albicans, Gram-negative bacteria Escherichia coli, Enterobacter aerogenes, Neisseria sicca, Stenotrophomonas maltophilia, Gram-positive bacteria Deinococcus radiodurans, Enterococcus faecium, Staphylococcus epidermidis, Streptococcus sanguinis, and vegetative and spore forms of Geobacillus stearothermophilus are discussed. A similar microbicidal effect after about one-minute exposure was observed in all vegetative forms of the microorganisms. Measurement in growth inhibition zones on a semisolid medium was used to determine the dependence of the microbicidal effect on exposure time and the distance between electrodes. Counting of colonies served to assess the microbicidal effect of the discharge on contaminated inert surfaces observable after more than 1 min exposure. Geobacillus stearothermophilus spores were found to have several times lower susceptibility to the action of the discharge and the microbicidal effect was observed only after an 8 min exposure. Reaction with the iodide reagent did not unambiguously demonstrate the difference between ozone and singlet oxygen as presumed active components of the corona. The area distribution of reactive oxygen species was determined; it was found to differ from the Wartburg law depending on exposure time. Qualitative evidence was obtained on the penetration of the reactive oxygen species into the semisolid medium.
Asunto(s)
Bacterias/crecimiento & desarrollo , Candida/crecimiento & desarrollo , Descontaminación , Electricidad , Descontaminación/métodosRESUMEN
The basis of photodynamic therapy (PDT) is the phototoxicity resulting from co-action of light, sensitizer and oxygen. In this study we demonstrate in vitro phototoxicity measurement on G361 cell lines using ZnTPPS(4) sensitizer bound to cyclodextrin hpbetaCD. We have proved its photodamage effect on cancer cell lines in the visible region of spectrum. We used the halogen lamp (24V/250W) as a source of radiation. After 24h incubation of cell cultures with 10 microM ZnTPPS(4) and 1mM cyclodextrine hpbetaCD, the cells were irradiated for 7.5 min at the total irradiation dose of 12.5 Jcm(-2). Analysis of DNA damage in the cell line after PDT was proved by comet assay and using inversion fluorescent microscope with image analysis. This treatment method gave rise to DNA damage. The used radiation dose of visible light in the absence of sensitizers does not induce DNA breaks in tumour cells. In conclusion, binding of ZnTPPS(4) sensitizer to cyclodextrin hpbetaCD may improve the efficacy of PDT for the treatment of malign melanoma.
Asunto(s)
Daño del ADN , ADN de Cadena Simple/efectos de los fármacos , Metaloporfirinas/farmacología , Fotoquimioterapia , Fármacos Fotosensibilizantes/farmacología , Línea Celular Tumoral , Ensayo Cometa , Humanos , Luz , Melanoma , Factores de Tiempo , beta-CiclodextrinasRESUMEN
We report the phototoxicity of meso-tetrakis(4-sulphonatophenyl)porphine (TPPS4) and zinc metallocomplex (ZnTPPS4) sensitizers in the presence or absence of 2-hydroxypropyl-beta-cyclodextrin (HP-beta-CD) on G361human melanoma cells. Morphological changes in cell cultures have been evaluated using inversion fluorescent microscope and image analysis. Viability of cells was determined by means of molecular probes for fluorescence microscopy (LIVE/DEAD kit- double staining with Calcein AM and Ethidium Homodimer). The quantitative changes of cell viability in relation to sensitizers concentrations and irradiation doses were proved by fluorometric measurement with fluoroscan Ascent. We found that the most effective sensitizer is ZnTPPS4 bound to HP-beta-CD, since the IC50 value was 12.5 g/ml at the dose of light radiation of 10 J/cm2.
Asunto(s)
Melanoma/tratamiento farmacológico , Fotoquimioterapia , Porfirinas/toxicidad , Fármacos Sensibilizantes a Radiaciones/toxicidad , Neoplasias Cutáneas/tratamiento farmacológico , Pruebas de Toxicidad/métodos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Relación Dosis-Respuesta a Droga , Humanos , Rayos Infrarrojos , Concentración 50 Inhibidora , Luz , Melanoma/patología , Melanoma/radioterapia , Microscopía Fluorescente , Fotoquimioterapia/métodos , Neoplasias Cutáneas/patología , Neoplasias Cutáneas/radioterapiaRESUMEN
We report the formation of host-guest complexes between water-soluble calix[n]arene-p-tetrasulfonates (n = 4, 6, 8) or 2-hydroxypropyl-cyclodextrins (alpha-, beta-, gamma-) and the tetratosylate salt of 5,10,15,20-tetrakis(4-N-methylpyridyl)porphyrin (TMPyP). The binding constants ranging between 10(2) and 10(5) M-1 were calculated from the absorption and fluorescence changes. Calix[4]arene-p-tetrasulfonate has a high binding affinity and forms with TMPyP a 1:1 complex, whereas other calixarenes bind two molecules of TMPyP. Electrostatic attraction is the dominating binding mode. Binding to calixarenes leads to a considerable decrease of the quantum yields of the triplet and excited singlet states and to shortening of the singlet and triplet lifetimes of TMPyP. The quenching mechanism is attributed to electron transfer between calixarene phenolates and excited TMPyP. Photoinduced electron transfer within a novel supramolecular complex calixarene/TMPyP (electron donor)/methyl viologen (electron acceptor) has been proven by absorption and fluorescence measurements. Electrostatic attraction between the cationic donor and cationic acceptor, on the one hand, and the anionic host, on the other, overcomes the electrostatic repulsion forces. In contrast, the interaction of cyclodextrin with TMPyP is hydrophobic in nature and only slightly influences the photophysical properties of TMPyP. The different behavior of TMPyP bound to either of the hosts has been assigned to the specific effects of the dominant binding modes, viz. the electrostatic attraction for calixarenes and the hydrophobic interactions for inclusion complexes with cyclodextrins.
Asunto(s)
Ciclodextrinas/química , Sustancias Macromoleculares , Porfirinas/química , Calixarenos , Electroquímica , Modelos Químicos , Estructura Molecular , Fotoquímica , Espectrometría de Fluorescencia , Espectrofotometría Atómica , Espectrofotometría UltravioletaRESUMEN
A rapid, sensitive and simple spectrophotometric method for the detection of 1O2 produced by photodynamic photosensitizers in slightly acid and air-saturated aqueous solutions has been developed. The method is based on the reaction of 1O2 (produced by photodynamic processes) with I- in the presence of ammonium molybdate as a catalyst. The reaction product I3-, proportional to 1O2, is followed spectrophotometrically at 355 nm. Several ways of avoiding interference with other oxidizing compounds, either present before or produced during the irradiation, are described.
Asunto(s)
Yoduros/análisis , Fármacos Fotosensibilizantes/análisis , Espectrofotometría/métodos , Humanos , Peróxido de Hidrógeno/química , Yoduros/química , Cinética , Molibdeno , Oxígeno/análisis , Oxígeno/química , Oxígeno SingleteRESUMEN
Under ischemic conditions, the excitatory amino acids (EAA), glutamate and aspartate, accumulate in the extracellular compartment of brain and, by excessive stimulation of EAA receptors, trigger excitotoxic degeneration of CNS neurons. Since glutamate and aspartate exert excitotoxic activity through both of the generally recognized classes of EAA receptors [N-methyl-D-aspartate (NMDA) and non-NMDA], it follows that both receptor classes may play a role in ischemic neuronal degeneration. Although several laboratories have reported that NMDA receptor antagonists confer protection in vivo against ischemic neuronal degeneration, very little is known about the ability of non-NMDA antagonists to confer such protection, a major reason being that non-NMDA antagonists that penetrate blood-brain barriers have not been available. In the present study, we examined the ability of NMDA or non-NMDA antagonists, either individually or in combination, to prevent neuronal degeneration in vivo in the adult rat retina rendered ischemic by dye/photothrombotic occlusion of retinal blood vessels. In this model, delivery of drugs to the ischemic tissue is assured by intravitreal administration. Intravitreal administration of the NMDA antagonist, MK-801, reduced the severity of ischemic damage approximately 50% (a ceiling effect that could not be increased by administering higher doses). The predominantly non-NMDA antagonist, CNQX, when administered in the highest dose permitted by its solubility limitations, provided equivocal (statistically nonsignificant) protection, but the two drugs combined provided greater than 80% protection.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Isquemia/patología , Degeneración Nerviosa/efectos de los fármacos , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Vasos Retinianos , Animales , Relación Dosis-Respuesta a Droga , Femenino , Isquemia/etiología , Isquemia/metabolismo , Ácido Quinurénico/análogos & derivados , Ácido Quinurénico/farmacología , Luz/efectos adversos , Ratas , Ratas Endogámicas , Oclusión de la Vena Retiniana/complicaciones , Oclusión de la Vena Retiniana/etiologíaRESUMEN
The endogenous excitotoxin, glutamate (Glu), acting at the N-methyl-aspartate (NMA) subtype of Glu receptor, is thought to play a major role in hypoxic/ischemic neuronal degeneration. In the present study, the sensitivities of the developing rat CNS to hypoxic/ischemic neuronal degeneration and to the neurotoxic action of NMA were compared at various postnatal ages. In the hypoxic/ischemic experiments, ischemia was produced by unilateral common carotid artery ligation and hypoxia by subjecting the pups to a partial vacuum. Keeping the duration of the hypobaric episode constant at 75 min for all age groups, we observed that the vulnerability of the immature brain to hypobaric/ischemic damage increased during the early neonatal period (days 2-4), reached a peak at day 6 and then diminished progressively with increasing age. In the second part of the study, NMA was microinjected unilaterally into the head of the caudate nucleus at various postnatal ages (2-80 d). In the early neonatal period (days 2-6), injections of relatively small doses of NMA (6-15 nmol) produced a dose-dependent widespread excitotoxic reaction throughout the forebrain with peak sensitivity being observed on day 6. The cytotoxic reaction to NMA was identical in appearance and time course to that induced by hypobaric/ischemic methods. With increasing age, the excitotoxic response to a given dose of NMA decreased progressively and the lesions became more strictly confined to the injection site. Cell populations most sensitive to NMA toxicity in the 2-10 d period closely correlated with those most vulnerable to hypoxia/ischemia, and sensitivity to both types of injury reached a peak at 6 d. These findings reinforce other evidence linking an excitotoxic mechanism and the NMA subtype of Glu receptor to hypoxic/ischemic brain damage and suggest that there may be a period during development when NMA receptors are hypersensitive to excitotoxic stimulation, thus rendering the neurons possessing such receptors hypervulnerable to hypoxic/ischemic damage.
Asunto(s)
Presión del Aire , Ácido Aspártico/análogos & derivados , Presión Atmosférica , Isquemia Encefálica/patología , Encéfalo/crecimiento & desarrollo , Animales , Animales Recién Nacidos , Ácido Aspártico/envenenamiento , Encéfalo/patología , Encéfalo/fisiología , Susceptibilidad a Enfermedades , Microinyecciones , N-Metilaspartato , Degeneración Nerviosa , Neuronas/efectos de los fármacos , Neuronas/patología , Ratas , Ratas EndogámicasRESUMEN
Here we describe a new model for inducing ischemic neuronal degeneration in the adult rat retina. Rose bengal dye was injected intravenously; then the retina was exposed to intense light which caused vascular photothrombosis resulting in acute degeneration of retinal neurons. By either light or electron microscopical criteria, the acute neurodegernative reaction was judged identical in pattern, cytopathological appearance, and time course to the excitotoxic type of reaction typically seen in the retina following exposure to exogenous glutamate. These results reinforce other accumulating evidence suggesting that ischemic CNS damage is mediated by glutamate or related excitotoxins. The advantages of this model of CNS ischemia include its noninvasiveness, ease of application, authentic simulation of vascular thrombosis, and accessibility for application of neuroprotective drugs.
Asunto(s)
Isquemia/etiología , Enfermedades de la Retina/complicaciones , Vasos Retinianos , Trombosis/complicaciones , Animales , Femenino , Isquemia/patología , Luz , Ratas , Ratas Endogámicas , Retina/patología , Enfermedades de la Retina/inducido químicamente , Enfermedades de la Retina/etiología , Rosa Bengala , Trombosis/inducido químicamente , Trombosis/etiologíaRESUMEN
We present a new animal model of perinatal hypoxic/ischemic brain damage and compare this type of brain damage with the excitotoxic type of damage previously described in the brains of infant rats and monkeys treated systemically with glutamate (Glu). Ten-d-old rats with unilateral occlusion of the common carotid artery were subjected to hypobaric conditions for 75 min and sacrificed 0-4 hr later for light and electron microscopic brain examination. The mortality rate was relatively low (12%), and brain damage was evident ipsilateral to the ligated carotid in 94% of surviving animals 4 hr after termination of the hypobaric event. Regions most frequently affected were the medial habenulum, dentate gyrus, caudate nucleus, frontoparietal neocortices, olfactory tubercle, and several thalamic nuclei. The acute cytopathological changes, primarily edematous degeneration of neuronal dendrites and cell bodies, evolved very rapidly, with some neurons manifesting end-stage necrosis at 0 hr (immediately after hypobaric exposure) and others developing such changes over a 1-4-hr period. We conclude that the neurodegenerative reaction induced in infant rat brain by hypoxia/ischemia is indistinguishable from the excitotoxic type of damage exogenous Glu is known to cause. Moreover, in a companion study (Olney et al., 1989) we show that MK-801, a powerful antagonist of the N-methyl-D-aspartate receptor complex (subtype of Glu receptor), protects against neuronal degeneration in this hypobaric/ischemic model. Our results reinforce other recent evidence suggesting that hypoxic/ischemic brain damage is mediated by endogenous Glu or related excitotoxins.
Asunto(s)
Presión Atmosférica , Isquemia Encefálica/patología , Encéfalo/patología , Glutamatos/farmacología , Animales , Animales Recién Nacidos , Encéfalo/efectos de los fármacos , Encéfalo/ultraestructura , Ácido Glutámico , Hipoxia/patología , Microscopía Electrónica , Ratas , Ratas EndogámicasRESUMEN
Accumulating evidence suggests that the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor may play an important role in hypoxic/ischemic (H/I) brain damage. Accordingly, it has been shown that the NMDA antagonist, MK-801, partially protects the infant rat brain against H/I damage. Here we show that reducing the body temperature of the infant rat also confers partial protection against H/I brain damage and that mild hypothermia plus MK-801 treatment provides total protection against such damage. Relevance of these findings to the prevention of perinatal brain damage in humans is discussed.
Asunto(s)
Anticonvulsivantes/uso terapéutico , Dibenzocicloheptenos/uso terapéutico , Hipotermia Inducida , Ataque Isquémico Transitorio/terapia , Receptores de Neurotransmisores/fisiología , Animales , Animales Recién Nacidos , Terapia Combinada , Maleato de Dizocilpina , Ataque Isquémico Transitorio/tratamiento farmacológico , Ataque Isquémico Transitorio/patología , Ratas , Ratas Endogámicas , Receptores de N-Metil-D-Aspartato , Receptores de Neurotransmisores/efectos de los fármacosRESUMEN
Recent evidence implicates the endogenous excitatory amino acids, glutamate (Glu) and aspartate, in hypoxic/ischemic neuronal degeneration. In a preceding article (Ikonomidou et al., 1989) we described a new model for studying hypoxic/ischemic neuronal degeneration in the infant rat brain that entails unilateral common carotid artery ligation followed by exposure to a partial vacuum for 75 min. Promising features of this model include a low mortality rate and high incidence of acute brain damage disseminated over numerous brain regions. In addition, there is a striking similarity between the type of cytopathology characterizing this model of hypoxic/ischemic neuronal degeneration and that which has been described in infant animals treated with Glu. MK-801 is a powerful antagonist of the N-methyl-D-aspartate (NMDA) receptor ionophore complex (a subtype of Glu receptor). In the present study, after unilateral carotid artery ligation was performed on 10-d-old rat pups, they were treated either with MK-801 (1 mg/kg i.p.) or saline 15 min before exposure to the hypobaric condition. MK-801 exerted a strong neuroprotective effect without serious side effects; the majority of saline control animals sustained severe brain damage, whereas the majority of MK-801-treated pups had no brain damage. These and other recent findings suggest that the NMDA receptor may play an important role in hypoxic/ischemic neuronal degeneration in the immature brain and provide hope that NMDA antagonists such as MK-801 may be effective in preventing such degeneration.
Asunto(s)
Animales Recién Nacidos/fisiología , Presión Atmosférica , Isquemia Encefálica/patología , Encéfalo/patología , Dibenzocicloheptenos/farmacología , Degeneración Nerviosa/efectos de los fármacos , Neuronas/patología , Animales , Temperatura Corporal , Encéfalo/efectos de los fármacos , Isquemia Encefálica/fisiopatología , Maleato de Dizocilpina , Femenino , Masculino , RatasRESUMEN
Autoradiographic techniques were used to label [3H]-adenosine and [3H]-cyclohexyladenosine accumulating cells in rabbit, mouse, and ground squirrel retinas. Immunohistochemical methods revealed the distribution of cells that stained for endogenous adenosine. Comparisons of these two markers revealed for all three species that the distribution of specific subpopulations of retinal cells that store or accumulate the purine nucleoside, adenosine, is similar. For all three species, cells localized in the ganglion cell layer accumulated adenosine and exhibited adenosine-like immunoreactivity (ALIR). A smaller proportion of cells localized in the inner nuclear layer were labeled for ALIR, while a larger proportion of cells in this layer accumulated adenosine. Subtle differences between species are presented. However, the general similarities of the distribution of these two putative purinergic markers supports the evidence that a discrete adenosinergic neurotransmitter/modulatory system is present in the retina.
Asunto(s)
Adenosina/metabolismo , Retina/metabolismo , Adenosina/análogos & derivados , Animales , Autorradiografía , Inmunohistoquímica , Ratones , Conejos , Retina/citología , Células Ganglionares de la Retina/metabolismo , Sciuridae , Distribución TisularRESUMEN
Rabbits were injected intravitreally with colchicine and the retinas were subsequently processed for simultaneous visualization of GAD-like and GABA-like immunoreactivity (LIR) at the light microscopical level. The use of specific antisera from unrelated species minimized the possibility of crossreactivity between the two markers. Colocalization of GABA-LIR and GAD-LIR was observed in approximately 32% of somata on the inner border of the inner nuclear layer (INL) and roughly 15% of the somata in the ganglion cell layer (GCL). All GAD-positive cells were double labeled whereas 15% of the GABA-LIR cells in the INL and 52% of those in the GCL were negative for GAD-LIR. Both markers were seen throughout the inner plexiform layer. In addition, processes coursing through the OPL, perhaps from interplexiform cells, and horizontal cell bodies were double-labeled with these GABAergic probes.
Asunto(s)
Glutamato Descarboxilasa/análisis , Retina/análisis , Ácido gamma-Aminobutírico/análisis , Animales , Reacciones Cruzadas , Técnica del Anticuerpo Fluorescente , Sueros Inmunes , Conejos , Células Ganglionares de la Retina/análisisRESUMEN
Rabbit antisera directed against gamma-amino butyric acid (GABA) conjugated to bovine serum albumin was used to localize neurons containing GABA-like immunoreactivity in the retinas of nine species of animals: human, cat, rabbit, rat, chicken, turtle, frog, mudpuppy and goldfish. The retinas of all species contained GABA-like labeling in several populations of amacrine cells in the inner nuclear layer, cells in the ganglion-cell layer that may include displaced amacrine cells and in fibers in the inner plexiform layer and in the optic nerve fiber layer. Labeled horizontal cells were found in cat and in all non-mammalian retinas. Labeled interplexiform cells were found in rat, rabbit, cat and human retinas. Labeled bipolar cells were restricted to frog and mudpuppy retinas. The distribution of anti-GABA is usually similar to that of anti-glutamic acid decarboxylase and neuronal [3H]GABA uptake, indicating good correspondence between these 'GABAergic' markers. However, several significant differences among these markers are discussed.
Asunto(s)
Retina/inmunología , Ácido gamma-Aminobutírico/inmunología , Animales , Gatos , Pollos , Carpa Dorada , Humanos , Necturus maculosus , Neuronas/inmunología , Nervio Óptico/inmunología , Conejos , Rana pipiens , Ratas , Células Ganglionares de la Retina/inmunología , TortugasRESUMEN
Rabbit retinas were double labeled to determine the degree of colocalization of glutamic-acid-decarboxylase-like immunoreactivity (GAD-like IR) and 3H-GABA uptake using light (LM) and electron microscopic (EM) autoradiography. Both GAD-like IR and 3H-GABA uptake were found in amacrine cell bodies in the inner nuclear layer (INL) as well as in cell bodies in the ganglion cell layer (GCL), and throughout the inner plexiform layer. GAD-like IR was found in 32% of the amacrine cells in the INL, 86% of which also showed 3H-GABA uptake; 3H-GABA uptake was observed in 38% of the amacrine cells. However, only 72% of these cells showed GAD-like IR. Labeled cells in the GCL were only 10-15% as common as similarly labeled cells in the INL. As in the INL, all GAD-positive cells in the GCL were double labeled, but only 53% of the cells taking up 3H-GABA were double labeled. We suggest that labeled cells in the GCL were ganglion cells rather than displaced amacrine cells. Cells, in both the INL and GCL, that showed 3H-GABA uptake but no GAD-like IR had a higher average grain density than double-labeled cells, indicating that uptake by these cells was specific. The relevance to GABAergic function of 3H-GABA uptake without an indication of GAD-like IR is yet to be determined. Statistical analysis at the EM level showed that one-third of the GAD-positive synaptic terminals of amacrine cells were double labeled after a 4-month exposure. Longer exposures at the EM level should reveal a higher percentage of GAD-positive terminals because at the LM level, one-half of the double-labeled cell bodies were "lightly" labeled with grains. The high degree of colocalization of GAD-like IR and 3H-GABA uptake suggests that both markers may be useful for labeling GABAergic neurons in the rabbit retina.