RESUMO
The effects of l-Arg, vitamin C (VC), and vitamin E (VE) on xanthine- (XO) and NAD(P)H-oxidase (NOX) activities, and nitric oxide (NO) availability of hypoxic broilers were evaluated. Chickens were kept in wire cages with free access to feed and water. One-day-old chicks were assigned to 1 of 3 diets: control (CTL; ME 3,200 kcal/kg, CP 23%), high Arg (HA; CTL + Arg 0.8%), or high Arg plus VE and VC (AEC; HA + 200 IU of VE/kg of feed + 500 mg of VC/L of water), and grown under hypobaric hypoxia (HYP) from d 7 to 30. A fourth group of birds was fed the CTL diet and grown under normoxia (CTL-NOR). At d 30, chickens were euthanized, their lungs fixed in vivo, excised, and processed for cyto- and histochemistry. The enzymes XO and NOX were localized and activities assessed histochemically and in lung homogenates. The NO depletion was assessed through nitrotyrosine immunocytochemistry colloidal gold particles (NTY). The XO and NOX localized in cell membranes and within vesicles of pulmonary vessel endothelial cells. The XO activity was higher in CTL-NOR birds (586 ± 43 reflectance units) than in both AEC-HYP (456 ± 39) and HA-HYP birds (394 ± 31), whereas CTL-HYP birds had the lowest XO activity (313 ± 27). The NO depletion was not affected by dietary or hypoxia conditions in clinically healthy birds; nevertheless, hypoxic birds that developed pulmonary hypertension had higher NTY levels (less NO, 145 ± 19) than hypoxic but clinically healthy birds (56 ± 11). Thus, the concurrent supplementation of Arg, VE, and VC restored XO activity without affecting NOX activity or NO availability. The dual role of XO, which produces superoxide and uric acid, may have buffered the effects of superoxide in broiler chickens grown under hypobaric hypoxia.
Assuntos
Arginina/farmacologia , Ácido Ascórbico/farmacologia , Proteínas Aviárias/metabolismo , Galinhas/metabolismo , NADPH Oxidases/metabolismo , Óxido Nítrico/metabolismo , Vitamina E/farmacologia , Xantina Oxidase/metabolismo , Ração Animal/análise , Animais , Arginina/administração & dosagem , Ácido Ascórbico/administração & dosagem , Biomarcadores/metabolismo , Dieta/veterinária , Suplementos Nutricionais/análise , Histocitoquímica/veterinária , Peróxido de Hidrogênio/metabolismo , Pulmão/metabolismo , Microscopia Confocal/veterinária , Estresse Oxidativo , Oxigênio/metabolismo , Tirosina/análogos & derivados , Tirosina/metabolismo , Vitamina E/administração & dosagemRESUMO
PURPOSE: Previous studies have suggested that disturbances in plasminogen activator inhibitor (PAI)-1 may be relevant to the development of diabetic microvascular complications. To determine whether overexpression of PAI-1 in cells of retinal microvasculature would result in a disease similar to that observed in diabetes, ocular tissue from transgenic mice that overexpress human PAI-1 were examined. METHODS: Transgenic mice were administered ZnSO4 (25 mM) in their water for up to 49 weeks to activate the metallothionein promoter and stimulate human PAI-1. Colloidal gold immunocytochemistry was used to quantify the human PAI-1 antigen at 7, 20, 34, and 49 weeks of ZnSO4 administration. Cross sections of retinal microvessels were examined by electron microscopy for changes in basement membrane (BM) thickness. Retinal digest preparations were examined by light microscopy for possible microangiopathy, including changes in endothelial cell-to-pericyte ratios. RESULTS: Human PAI-1 immunoreactivity was detected throughout the retinal capillaries of transgenic mice receiving zinc and increased significantly (P < 0.001) after 20 to 49 weeks of ZnSO4 administration compared with age-matched transgenic control mice. At 20 and 49 weeks, retinal capillaries of transgenic mice that received zinc showed significantly thickened BMs compared with control animals (P < 0.001). Moreover, wholemounts of the retinal vasculature from PAI-1 transgenic mice demonstrated an increased endothelial cell-to-pericyte ratio. CONCLUSIONS: PAI-1 overexpression in retinal microvasculature leads to retinal disease similar to that observed in diabetic retinopathy.
Assuntos
Camundongos Transgênicos , Inibidor 1 de Ativador de Plasminogênio/biossíntese , Vasos Retinianos/metabolismo , Inibidores de Serina Proteinase/biossíntese , Animais , Membrana Basal/metabolismo , Membrana Basal/ultraestrutura , Capilares , Retinopatia Diabética/metabolismo , Retinopatia Diabética/patologia , Endotélio Vascular/metabolismo , Endotélio Vascular/ultraestrutura , Feminino , Masculino , Camundongos , Microscopia Imunoeletrônica , Pericitos/metabolismo , Pericitos/ultraestrutura , Inibidor 1 de Ativador de Plasminogênio/genética , Vasos Retinianos/ultraestrutura , Inibidores de Serina Proteinase/genética , Sulfato de Zinco/administração & dosagemRESUMO
Hyperglycemia in diabetes induces increased levels of hydrogen peroxide (H2O2), a reactive oxygen species generated by reduced nicotinamide adenine dinucleotide (NADH) oxidase. Nontoxic levels of H2O2 increase endothelial cell permeability. Using a model of non-insulin-dependent diabetes, the BBZ/Wor rat, we investigated retinal levels of H2O2, vascular endothelial growth factor (VEGF) and its receptors, VEGF-R1 and VEGF-R2 by transmission electron microscopy at sites of the blood-retinal barrier (BRB). H2O2 localization was done by the cerium NADH oxidase method, and extravasation of endogenous serum albumin was used to document disruption of the BRB. Higher levels of H2O2 were detected in blood vessels of diabetic (78.7 +/- 4.84%) as compared with vessels from nondiabetic rats (39.0 +/- 4.47%). VEGF immunoreactivity was statistically higher in the inner BRB (24.67 +/- 0.33 colloidal gold particles/63 microm2 vs. 21.52 +/- 0.43 colloidal gold particles/63 microm2, p = .0001) and outer BRB (42.56 +/- 0.45 colloidal gold particles/63 microm2 vs. 15.51 +/- 0.51 colloidal gold particles/63 microm2, p = .0001) of diabetic rats as compared with age matched nondiabetic control rats. VEGF-R1 immunoreactivity was significantly higher in diabetic retinas in both the inner BRB (21.66 +/- 0.75 colloidal gold particles/63 microm2 vs. 12.69 +/- 0.61 colloidal gold particles/63 microm2, p = .0001) and outer BRB (22.76 +/- 2.36 colloidal gold particles/63 microm2 vs. 8.53 +/- 2.67 colloidal gold particles/63 microm2, p = .0013). VEGF-R2 was statistically higher in the inner BRB (8.97 +/- 0.57 colloidal gold particles/63 microm2 versus 7.03 +/- 0.65 colloidal gold particles/63 microm2, p = .0419) but not in the outer BRB (29.42 +/- 1.25 colloidal gold particles/63 microm2 vs. 28.07 +/- 1.42 colloidal gold particles/63 microm2, p = .4889). H2O2 levels correlated with increased VEGF (correlation coefficient = 0.82, p = .001) in this model of nonproliferative diabetic retinopathy. These results support that hyperglycemia is one factor that induces retinal endothelial cells in vivo to increase H2O2 via NADH oxidase and stimulates increases in VEGF resulting in disruption of the BRB.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Retinopatia Diabética/metabolismo , Modelos Animais de Doenças , Fatores de Crescimento Endotelial/metabolismo , Proteínas do Olho/metabolismo , Peróxido de Hidrogênio/metabolismo , Linfocinas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Ratos Mutantes/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Fatores de Crescimento/metabolismo , Retina/metabolismo , Animais , Glicemia/análise , Barreira Hematorretiniana , Hemoglobinas Glicadas/análise , Masculino , Ratos , Ratos Endogâmicos , Espécies Reativas de Oxigênio/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular , Fator A de Crescimento do Endotélio Vascular , Receptor 1 de Fatores de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
This morphological study demonstrates a role for endothelial cells in generating reactive oxygen species in early stages of retinopathy in the BBZ/Wor rat, an obese, noninsulin dependent model of diabetes. Hyperglycemia induced pseudohypoxia results in an imbalance in cytosolic NADH/NAD+. In the oxygen-rich environment of the retina, NADH oxidase generates superoxide radical which is dismutated to hydrogen peroxide. Localization of hydrogen peroxide by the cerium NADH oxidase enzyme activity cytochemical localization technique shows a statistically significant increase of peroxide localization in the central retina of diabetic rats as compared to age-matched, nondiabetic controls. Endothelial cell dysfunction, indicated by leakage of endogenous serum albumin, coincided with areas of NADH oxidase activity localization. In diabetic rats there are increased levels of fibronectin in areas of hydrogen peroxide localization. This in vivo, morphological study is the first demonstration of oxidative injury and endothelial cell dysfunction in the retina of a spontaneous, noninsulin dependent model of diabetes.
Assuntos
Diabetes Mellitus Experimental/fisiopatologia , Diabetes Mellitus Tipo 2/fisiopatologia , Diabetes Mellitus/fisiopatologia , Complexos Multienzimáticos/metabolismo , NADH NADPH Oxirredutases/metabolismo , Obesidade , Estresse Oxidativo/fisiologia , Retina/enzimologia , Animais , Diabetes Mellitus/enzimologia , Diabetes Mellitus Experimental/enzimologia , Diabetes Mellitus Tipo 2/enzimologia , Endotélio Vascular/fisiologia , Radicais Livres , Histocitoquímica , Imuno-Histoquímica , Masculino , Ratos , Ratos EndogâmicosRESUMO
Human retinal endothelial cell (HREC) cultures of diabetic and non-diabetic origin were examined for the production of insulin-like growth factor I (IGF-I), IGF-I receptor and IGF-binding proteins (IGFBPs) using colloidal gold quantitative immunocytochemistry and quantitative reverse transcriptase-polymerase chain reaction (QRT-PCR). The levels of immunoreactivity for IGF-I receptor and for four IGFBPs (IGFBP-1, -2, -3 and -5) were significantly increased in diabetic HREC cultures. Moreover, diabetic HREC cultures showed significantly less immunoreactivity for IGF-I and for IGFBP-4 as compared to non-diabetic HREC cultures. Message levels for IGF-I decreased two-fold in diabetic HREC and correlated with protein levels. Message levels for IGFBP-1, -2 and -5 increased 1.5-, 1.7- and 1.6-fold, respectively, in diabetic HREC and correlated with protein levels. However, the protein levels for IGF-I receptor and IGFBP-3 and -4 did not correlate with mRNA levels. There were no differences in mRNA levels for IGF-I receptor and IGFBP-3 and -4 between diabetic and non-diabetic HREC cultures, suggesting a post-transcriptional regulation of IGF-I receptor and the two IGFBPs. The net effect, however, supports enhanced IGF-I action in HREC cultures of diabetic origin which is an important cellular event in diabetic retinopathy.
Assuntos
Diabetes Mellitus/metabolismo , Endotélio/metabolismo , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Receptor IGF Tipo 1/metabolismo , Retina/metabolismo , Células Cultivadas , Diabetes Mellitus/patologia , Retinopatia Diabética/metabolismo , Endotélio/citologia , Endotélio/ultraestrutura , Feminino , Regulação da Expressão Gênica , Humanos , Imuno-Histoquímica , Hibridização In Situ , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/genética , Fator de Crescimento Insulin-Like I/genética , Masculino , Microscopia Imunoeletrônica , Pessoa de Meia-Idade , RNA Mensageiro/metabolismo , Receptor IGF Tipo 1/genética , Retina/citologia , Retina/ultraestrutura , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The molecular and cellular processes that induce rapid atherosclerotic plaque progression in patients with unstable angina and initiate restenosis following coronary interventional procedures are uncertain. We examined primary (de novo) and restenotic lesions retrieved at the time of directional coronary atherectomy for expression of insulin-like-growth factor-I (IGF-I). IGF-I receptor, and five IGF binding proteins (IGFBPs), IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, and IGFBP-5 in smooth muscle cells (SMCs) using colloidal gold immunocytochemistry. IGF-1, its receptor and binding proteins were not detected in SMCs of normal coronary arteries. IGF-I localized primarily in synthetic smooth muscle cells (sSMCs) in both de novo and restenotic plaques. IGF-I receptor localized on sSMCs and their processes and colocalized with IGF-I. Although morphometric analysis of IGF-I and IGF-I receptor immunoreactivity in sSMCs of de novo and restenotic lesions showed comparable levels of IGF-I (3.2 +/- 1.0 and 2.9 +/- 0.9, respectively). IGF-I receptor was significantly higher in de novo lesions as compared to restenotic lesions (10.7 +/- 2.5 and 4.2 +/- 1.3, P < 0.05, respectively). IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4 and IGFBP-5 localized in the cytoplasm of sSMCs and in the extracellular matrix. Quantitative reverse transcription polymerase chain reaction (QRT-PCR) performed on de novo atherectomy specimens identified mRNA for IGF-I, IGF-I receptor, IGFBP-1, IGFBP-2, IGFBP-4, IGFBP-5 levels and detected mRNA for IGFBP-3. The expression of IGF-I, IGF-I receptor, and IGFBPs in atherectomy plaques suggests that the development of coronary obstructive lesions may be a result of changes in the IGF system.
Assuntos
Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Receptor IGF Tipo 1/metabolismo , Aterectomia Coronária , Doença da Artéria Coronariana/metabolismo , Vasos Coronários/química , Vasos Coronários/metabolismo , Eletroforese em Gel de Ágar , Regulação da Expressão Gênica/genética , Coloide de Ouro/imunologia , Humanos , Microscopia Imunoeletrônica , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , RNA Mensageiro/metabolismoRESUMO
Plasminogen activator inhibitor-1 is secreted bidirectionally by endothelial cells, acts as the primary regulator of fibrinolysis and as a key modulator of extracellular matrix proteolysis. Elevated serum levels of plasminogen activator inhibitor-1 are observed in serum of diabetic individuals. We investigated whether plasminogen activator inhibitor-1 is overexpressed in capillaries of diabetic donors with non-proliferative retinopathy compared to non-diabetic donors. We also assessed plasminogen activator inhibitor-1 expression in an animal model of retinopathy induced by exposing rabbit retinas to insulin-like growth factor-I. Colloidal gold immunocytochemistry was used to quantify plasminogen activator-1 antigen in donor retinas from diabetic subjects (n = 10) and control subjects (n = 10). This technique was also used to examine expression of plasminogen activator inhibitor-1 for correlation with retinal changes in the insulin-like growth factor-I-induced retinopathy model (n = 14). Plasminogen activator inhibitor-1 immunoreactivity was significantly increased in the retinas of all diabetic subjects as compared to controls. In the rabbit model, the expression of plasminogen activator inhibitor-1 immunoreactivity correlated with pathological retinal changes. In both the diabetic human and insulin-like growth factor-I-injected rabbit, overproduction of plasminogen activator inhibitor-1 was seen within the lumen of capillaries, within the cytoplasm of endothelial cells and in the basement membrane and extracellular matrix surrounding these capillaries. Minimal plasminogen activator inhibitor-1 was detected in the retinas of non-diabetics and in control rabbits injected with either heat-inactivated insulin-like growth factor-I or balanced salt solution. These studies support the conclusion that plasminogen activator inhibitor-1 is overexpressed in the retinal capillaries of diabetics with non-proliferative diabetic retinopathy and in rabbits with insulin-like growth factor-I-induced retinopathy.
Assuntos
Retinopatia Diabética/metabolismo , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Adulto , Idoso , Animais , Membrana Basal/metabolismo , Capilares/metabolismo , Retinopatia Diabética/patologia , Matriz Extracelular/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Masculino , Microscopia Eletrônica , Pessoa de Meia-Idade , Inibidor 1 de Ativador de Plasminogênio/análise , CoelhosRESUMO
In this study, we demonstrate, for the first time, the localization of insulin-like growth factor I (IGF-I) in de novo and restenotic human coronary atherectomy plaques by using immunocytochemical techniques. Smooth muscle cells (SMCs) exhibiting the synthetic phenotype contained a statistically significant higher concentration of IGF-I than SMCs of the contractile phenotype or SMCs from normal coronary arteries. In addition, we provide data to suggest that the long-acting somatostatin analogues octreotide and angiopeptin inhibit IGF-I- and basic fibroblast growth factor (b-FGF)- induced human coronary artery SMC proliferation. Platelet-derived growth factor (PDGF)-stimulated cultures were minimally affected by the addition of octreotide but were significantly inhibited by angiopeptin. All three growth factors stimulated SMC migration in a dose-dependent manner. The somatostatin analogues tested had no effect on growth factor-stimulated SMC migration. Our data suggest that by reducing SMC proliferation, somatostatin analogues may have clinical usefulness in reducing the high incidence of restenosis observed after percutaneous transluminal coronary artery interventions.
Assuntos
Doença da Artéria Coronariana/patologia , Vasos Coronários/efeitos dos fármacos , Fator de Crescimento Insulin-Like I/análise , Músculo Liso Vascular/efeitos dos fármacos , Octreotida/farmacologia , Oligopeptídeos/farmacologia , Somatostatina/análogos & derivados , Adulto , Angioplastia Coronária com Balão , Divisão Celular/efeitos dos fármacos , Constrição Patológica/tratamento farmacológico , Vasos Coronários/química , Vasos Coronários/citologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Humanos , Músculo Liso Vascular/química , Músculo Liso Vascular/citologia , Peptídeos Cíclicos , Fator de Crescimento Derivado de Plaquetas/farmacologia , Recidiva , Somatostatina/farmacologiaAssuntos
Córnea/efeitos dos fármacos , Endotélio Vascular/fisiologia , Fator de Crescimento Insulin-Like I/farmacologia , Neovascularização Patológica , Vasos Retinianos/efeitos dos fármacos , Animais , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Quimiotaxia/efeitos dos fármacos , Córnea/patologia , Preparações de Ação Retardada , Implantes de Medicamento , Endotélio Vascular/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/farmacologia , Humanos , Técnicas In Vitro , Coelhos , Proteínas Recombinantes/farmacologia , Vasos Retinianos/citologia , Vasos Retinianos/patologiaRESUMO
The release of growth factors from ischaemic retina has been hypothesized as the central stimulus for retinal neovascularization in proliferative diabetic retinopathy. Two of the growth factors implicated are insulin-like growth factor-I and basic fibroblast growth factor. We examined the effect of insulin-like growth factor-I on in vivo neovascularization using the established angiogenic model of the rabbit cornea (n = 30), and also compared the effects of insulin-like growth factor-I and basic fibroblast growth factor using two new in vivo systems. Either supraphysiologic concentrations of each growth factor (600 micrograms) were injected intravitreally into pigmented rabbits (n = 21) or porous polyfluorotetraethylene chambers filled with an emulsion containing collagen and growth factor (500 ng) were placed on the retina surface (n = 8). Our results demonstrate that when insulin-like growth factor-I was implanted together with a slow release carrier into the pocket of the normally avascular cornea, insulin-like growth factor-I (10 micrograms/pellet) induced angiogenesis in all rabbits. This degree of angiogenesis was comparable to that previously shown for basic fibroblast growth factor. For the intravitreal studies, the fibrotic component was greater in the basic fibroblast growth factor injected eyes, whereas the vascular component was accentuated in the eyes injected with insulin-like growth factor-I. Light and electron microscopy demonstrated areas of vascular proliferation in both groups. Porous polyfluorotetraethylene chamber studies with insulin-like growth factor-I and basic fibroblast growth factor demonstrated vascular proliferation in the vicinity of the chamber similar to the intravitreal injected eyes, but to a lesser degree than the injected eyes. Our experiments overall support the angiogenic potential of both insulin-like growth factor-I and basic fibroblast growth factor and support distinct but complimentary roles for each growth factor in the pathogenesis of proliferative diabetic retinopathy.
Assuntos
Córnea/patologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Fator de Crescimento Insulin-Like I/farmacologia , Neovascularização Patológica , Retina/patologia , Vasos Retinianos/efeitos dos fármacos , Animais , Córnea/irrigação sanguínea , Córnea/efeitos dos fármacos , Preparações de Ação Retardada , Microscopia Eletrônica , Nervo Óptico/irrigação sanguínea , Polivinil , Coelhos , Retina/efeitos dos fármacos , Vasos Retinianos/patologia , Vasos Retinianos/ultraestruturaRESUMO
In order to determine the role of H2O2 in demyelination of the optic nerve, serial quantitative analysis of H2O2-derived cerium perhydroxide reaction product particles was obtained by computerized digitization of electron micrographs of the myelinated retrobulbar optic nerve, unmyelinated optic nerve head, and the optic nerve sheath of guinea pigs sensitized for experimental allergic encephalomyelitis (EAE) and euthanized 3-14 days later. We found that cerium perhydroxide reaction product particles were greatest in the myelinated optic nerve 3 days after antigenic sensitization, but at this focus decreased 7-14 days after antigenic sensitization. Reaction product accumulated in the unmyelinated optic nerve head and optic nerve sheath 3-14 days after sensitization. These results in the myelinated optic nerve suggest H2O2 consumption results in peroxidation of myelin lipid as demyelination proceeds 7-14 days after antigenic sensitization. Hydrogen peroxide accumulation in the optic nerve head and the optic nerve sheath appears to provide a reservoir for diffusion of H2O2 into the retrobulbar optic nerve and adjacent perineural nerve, contributing to the frequent predilection for optic nerve involvement in EAE and perhaps in multiple sclerosis.
Assuntos
Encefalomielite Autoimune Experimental/metabolismo , Peróxido de Hidrogênio/metabolismo , Neurite Óptica/metabolismo , Animais , Encefalomielite Autoimune Experimental/patologia , Radicais Livres , Cobaias , Histocitoquímica , Processamento de Imagem Assistida por Computador , Peroxidação de Lipídeos , Bainha de Mielina/metabolismo , Bainha de Mielina/ultraestrutura , Disco Óptico/metabolismo , Disco Óptico/ultraestrutura , Nervo Óptico/metabolismo , Nervo Óptico/ultraestrutura , Neurite Óptica/patologiaRESUMO
To provide magnetic resonance imaging (MRI)-ultrastructural correlations of demyelinating lesions of the intraorbital optic nerve, the authors performed gadolinium-enhanced/T2-weighted fat-suppressed MRI and transmission electron microscopy of the optic nerves of animals with experimental allergic encephalomyelitis. Gadolinium enhancement of the optic nerve adjacent to the globe was seen on fat-suppressed T1-weighted MRI as early as 3 days after antigenic sensitization, increased in severity involving longer segments of nerve at 10 to 14 days, and persisted at 30 days. Gadolinium enhancement preceded T2-weighted signal aberrations. Ultrastructural evaluation of the intraorbital nerve revealed: (1) expansion of the extracellular space and inflammatory infiltrate that correlated with the intensity of gadolinium enhancement; (2) the degree of demyelination correlated with T2-weighted signal aberrations; (3) as deduced from gadolinium enhancement and T2 signal aberrations, breakdown of the blood-brain barrier preceded widespread demyelination; (4) lesions appeared to start at the optic nerve insertion into the globe and then progress toward the orbital apex.
Assuntos
Encefalomielite Autoimune Experimental/patologia , Imageamento por Ressonância Magnética/métodos , Neurite Óptica/patologia , Tecido Adiposo/ultraestrutura , Animais , Meios de Contraste , Combinação de Medicamentos , Gadolínio , Gadolínio DTPA , Cobaias , Meglumina , Nervo Óptico/ultraestrutura , Órbita , Compostos Organometálicos , Ácido PentéticoRESUMO
The spectra of myelin sheath thickness and g ratio (axon diameter/fibre diameter) of guinea pig optic nerves for 8 animals with acute experimental allergic encephalomyelitis (EAE) were compared with those for 6 normal animals. The mean myelin sheath thickness of 0.12 microns for the animals with EAE was significantly lower than the value of 0.16 microns for the normal animals. Since fibre diameter comprises axon diameter plus the thickness of its surrounding myelin sheath, a reduction in mean fibre diameter from 1.52 microns in normals to 1.20 microns in EAE was expected, but it was surprising to find that a mean g ratio of 0.85 obtained for normal nerves was not substantially different from a value of 0.86 for demyelinated optic nerves. A decrease in the mean axon diameter of 1.24 microns for normal animals to 0.94 microns for those with EAE tended to offset the decrease in mean myelin sheath thickness and contributed to the relative stability of the g ratio with acute demyelination. Our results showing reduction in axonal calibre and myelin sheath thickness may offer an explanation for apparent discrepancies between electrophysiological delays in conduction characteristics of experimental and, if not the result of a maturational effect on myelination, human primary demyelinating disorders associated with the scant histopathological demyelination of initial attacks of EAE and the visually asymptomatic patients with multiple sclerosis.
Assuntos
Encefalomielite Autoimune Experimental/patologia , Bainha de Mielina/ultraestrutura , Fibras Nervosas Mielinizadas/ultraestrutura , Nervo Óptico/ultraestrutura , Animais , Feminino , Cobaias , Microscopia Eletrônica , Nervo Óptico/patologia , Valores de ReferênciaRESUMO
The association of reactive oxygen species to altered permeability of the blood-brain barrier in acute experimental encephalomyelitis was investigated by ultrastructural cytochemical localization of hydrogen peroxide (H2O2) to sites in the optic nerve previously identified by extravasation of intravascular horseradish peroxidase. Using a modified cerium method, we found electron-dense cerium-derived H2O2 reaction product was localized to the perivascular space at the lamina retinalis, lamina choroidalis, and lamina scleralis. In the optic nerve head, electron-dense reaction product was observed in the presence of intravascular leukocytes, although adjacent perivascular and interstitial inflammatory cells at this site were scant. In the myelinated retrobulbar optic nerve, cerium-derived H2O2 reaction product was seen in the intravascular space of blood vessels and surrounding perivascular and interstitial foci of inflammatory cells. Reaction product was also observed in the extracellular space adjacent to the plasmalemma of axons and glial cells in the optic nerve head and retrobulbar nerve. The perivascular and intravascular distribution of cerium-derived reaction product suggests that H2O2 may play a role in the pathogenesis of altered vascular permeability in experimental optic neuritis and supports our previous observations of suppression of blood-brain barrier permeability by detoxification of H2O2 with the exogenous administration of antioxidant enzymes.
Assuntos
Peróxido de Hidrogênio/metabolismo , Neurite Óptica/metabolismo , Animais , Encefalomielite Autoimune Experimental/complicações , Encefalomielite Autoimune Experimental/metabolismo , Cobaias , Disco Óptico/metabolismo , Disco Óptico/ultraestrutura , Neurite Óptica/etiologia , Neurite Óptica/patologiaRESUMO
Magnetic resonance imaging (MRI) with gadolinium-diethylenetriaminepentaacetic acid (Gd-DTPA) was used to investigate disruption of the blood-optic nerve barrier associated with acute autoimmune demyelination. Leakage of Gd-DTPA was seen in the optic nerves and optic chiasm of adult guinea pigs sensitized for acute experimental allergic encephalomyelitis, but not in normal unsensitized animals. This finding occurred as early as 5 to 8 days after antigenic sensitization with the myelin emulsion and before the onset of paralysis or ataxia. Pathologic examination at this early stage of experimental allergic encephalomyelitis showed an absence of demyelination in the optic nerves and optic chiasm, although scant perivascular foci of inflammatory cells were seen. Leakage of Gd-DTPA in the optic nerve before demyelination of this white matter tract illustrates that increased permeability of the blood-optic nerve barrier is an early, if not the initial, event in autoimmune demyelination, and the optic nerve is a common site of central nervous system involvement during the initial phase of acute experimental allergic encephalomyelitis. Findings in this animal model appear comparable with the results of MRI with Gd-DTPA in patients with optic neuritis, and they suggest that disruption of the blood-optic nerve barrier is a common denominator for both disorders of primary demyelination.
Assuntos
Aumento da Imagem/métodos , Imageamento por Ressonância Magnética , Neurite Óptica/diagnóstico , Compostos Organometálicos , Ácido Pentético , Animais , Doenças Desmielinizantes/patologia , Modelos Animais de Doenças , Gadolínio DTPA , Cobaias , Quiasma Óptico/patologia , Nervo Óptico/patologia , Neurite Óptica/patologia , Compostos Organometálicos/farmacocinética , Ácido Pentético/farmacocinética , PermeabilidadeRESUMO
We studied the effect of antioxidant enzymes on the loss of integrity of the blood-brain barrier in the optic nerves of strain-13 guinea pigs with chronic experimental allergic encephalomyelitis, a demyelinating disorder with neurologic and histopathologic characteristics similar to multiple sclerosis. Animals with experimental allergic encephalomyelitis received daily intraperitoneal injections of either preservative-free saline (group 1), catalase (group 2), or glutathione peroxidase (group 3) for 2.2 months after the onset of appendicular paralysis. Following intravascular administration, extravascular leakage of horseradish peroxidase was histopathologically graded as mild, moderate, or severe within the optic nerve head and myelinated retrolaminar nerve. Severe extravasation of horseradish peroxidase was exclusive to group 1, in addition to moderate and mild leakage. In groups 2 and 3, leakage of horseradish peroxidase was infrequent, and when detected, it was graded as mild. Detoxification of hydrogen peroxide with catalase and glutathione peroxidase substantially reduced horseradish peroxidase leakage in experimental optic neuritis, suggesting a role for hydrogen peroxide and its reactive by-products in the pathogenesis of increased vascular permeability of the blood-brain barrier in experimental allergic encephalomyelitis.
Assuntos
Barreira Hematoencefálica/efeitos dos fármacos , Catalase/farmacologia , Glutationa Peroxidase/farmacologia , Neurite Óptica/metabolismo , Animais , Permeabilidade Capilar/efeitos dos fármacos , Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/patologia , Cobaias , Peroxidase do Rábano SilvestreRESUMO
The spectra of fiber and axon diameter, myelin sheath thickness, fiber density, and g ratio of the optic nerve were analyzed for the strain-13 guinea pig, an animal extensively utilized in the investigation of experimental disorders of demyelination. Our detailed analytical study of the normal guinea pig optic nerve provides the basis for comparison to disease states and the morphology of other species. As in the rat, mouse, and chipmunk, fiber diameters in the guinea pig were unimodal, but dissimilar to the trimodal fiber spectra of the cat and primate. The predominance of medium-sized fibers (0.80-2.00 microns), common to most species, contributed to the larger mean fiber diameter (1.45 microns) of the guinea pig optic nerve, in which small fibers (0.50 microns or less) were infrequent and fibers larger than 5.00 microns in diameter, seen in the cat and primate, were absent. While myelin sheath thickness increased with axon diameter in the guinea pig, as in other species, a g ratio of 0.81 in the guinea pig was greater than in most mammals. Since conduction velocity is dependent on axon size, as well as myelin properties, the relatively larger mean axon diameter of the guinea pig optic nerve (1.18 microns) may compensate for the decrease in its myelination.
Assuntos
Cobaias/anatomia & histologia , Processamento de Imagem Assistida por Computador , Fibras Nervosas Mielinizadas/ultraestrutura , Nervo Óptico/citologia , Animais , Contagem de Células , Microscopia Eletrônica , Bainha de Mielina/ultraestruturaRESUMO
Detoxification of hydrogen peroxide by the antioxidant enzyme catalase suppressed the neurologic manifestations of acute experimental allergic encephalomyelitis (EAE) and prevented death of treated adult strain-13 guinea pigs. The oxygen radical scavenger superoxide dismutase (SOD) delayed the onset of paralysis by one day, but did not prevent death from encephalomyelitis common to most of this group and all untreated animals. Histopathologic analysis of the optic nerves confirmed a statistically significant reduction in demyelination with catalase treatment, but not with SOD. Hydrogen peroxide, and/or its conversion products, discharged by phagocytic mononuclear cells, may play a role in the pathogenesis of demyelination in experimental optic neuritis.
Assuntos
Antioxidantes/uso terapêutico , Neurite Autoimune Experimental/complicações , Animais , Catalase/farmacologia , Doenças Desmielinizantes/tratamento farmacológico , Doenças Desmielinizantes/patologia , Cobaias , Injeções Intraperitoneais , Neurite Autoimune Experimental/patologia , Nervo Óptico/patologia , Superóxido Dismutase/farmacologiaRESUMO
In order to determine if axonal transport changes in chronic experimental allergic encephalomyelitis (EAE) were due to blockade or increased discharge of fast transported proteins from the inner retina, we examined the presence of pulse labeled proteins in autoradiograms of the optic nerve head, retinal ganglion cell and nerve fiber layers of juvenile strain-13 guinea pigs with chronic EAE and normal controls. Quantitative analysis of silver grains, performed six and twenty-four hours following the intravitreal injection of tritiated leucine, showed a decrease in inner retinal radioactivity in those with EAE, whereas no difference was detected between the two groups after three days. Grain counts within the optic nerve heads of guinea pigs with EAE were reduced at all time intervals studied. These results are consistent with an increase in discharge of fast transported proteins from retinal ganglion cells into optic nerve axons and support our previous observations of increased radioactivity at the foci of optic nerve demyelination.
Assuntos
Proteínas do Olho/análise , Neurite Óptica/metabolismo , Retina/análise , Animais , Autorradiografia , Encefalomielite Autoimune Experimental/metabolismo , Cobaias , Fibras Nervosas/análise , Disco Óptico/análise , Células Ganglionares da Retina/análise , PrataRESUMO
In order to determine if changes in axonal transport were different in adult animals with acute experimental allergic encephalomyelitis (EAE), in comparison to juvenile animals with chronic EAE, the effects of this acute demyelinating disorder on axonal transport were examined in the optic nerves of adult strain-13 guinea pigs. Utilizing autoradiographic analysis of silver grain counts, both the fast and slow components of orthograde transport were studied at intervals of thirty minutes, three hours, one day and three days after tritiated leucine injection into the vitreous cavity. In order to determine the contribution of fiber loss in acute EAE, optic nerve fiber density was analyzed from electron micrographs of normal and demyelinated nerves. Animals with acute EAE had a decrease in radioactivity at the lamina retinalis and lamina choroidalis after thirty minutes and three hours, and at the lamina scleralis and foci of demyelination after one and three days. A 16% loss of fibers did not account for as much as a 74% reduction in radioactivity with acute EAE. The global reductions in axonal transport observed in acute EAE animals may contribute to their progressive deterioration and eventual demise by lack of delivery of tubulo-vesicular materials for synaptic transmission, axolemmal proteins for electrogenesis and neurofilamentary components of the cytoskeleton. Moreover, they are unlike the increase of fast axonal transport associated with recovery of physiologic function characteristic of animals with the chronic form of the disease.