RESUMO
PURPOSE: Almost every female classic galactosemia patient develops primary ovarian insufficiency (POI). The unique pathophysiology of classic galactosemia, with a severely reduced follicle pool at an early age, requires a new therapeutic approach. This study evaluated the effect of dehydroepiandrosterone (DHEA) on ovarian tissue in a galactose-induced POI rat model. METHODS: Pregnant rats were fed with either a normal or a 35% galactose-containing diet from day 3 of conception continuing through weaning of the litters. Galactose-exposed female offspring were further divided into 5 groups on PND21. The first group received no application. Treatment groups were fed orally by gavage once daily with sesame oil (group 2), or DHEA at doses of 0.1 mg/kg (group 3), 1 mg/kg (group 4) or 10 mg/kg (group 5) until PND70. Fertility rates of mothers with galactosemia, body weights (BWs), and ovarian weights of the litters from PND21 to PND70 were recorded. Ovarian follicle count, immunohistochemistry for proliferation and apoptosis marker expressions and TUNEL for cell death assessment were performed in offspring ovaries. RESULTS: Decreased fertility, ovarian/body weights were observed under galactosemic conditions, together with decreased follicle number and increased atresia. Improved postnatal development, primordial follicle recruitment and follicular growth were observed after DHEA treatment. After DHEA treatment, the expression of Ki67 protein was found to be increased; elevated expression of cleaved-caspase-3 under galactosemia was found to be reduced. CONCLUSIONS: Our data suggests that DHEA treatment may be a potentially useful clinical therapy to improve ovarian ageing in women with POI-induced by galactosemia.
Assuntos
Envelhecimento/efeitos dos fármacos , Desidroepiandrosterona/farmacologia , Galactosemias/dietoterapia , Insuficiência Ovariana Primária/dietoterapia , Envelhecimento/genética , Animais , Suplementos Nutricionais , Modelos Animais de Doenças , Feminino , Galactose/toxicidade , Galactosemias/induzido quimicamente , Galactosemias/complicações , Galactosemias/patologia , Humanos , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/patologia , Gravidez , Insuficiência Ovariana Primária/induzido quimicamente , Insuficiência Ovariana Primária/patologia , RatosRESUMO
AIMS/HYPOTHESIS: Osmotic and oxidative stress is associated with the progression and advancement of diabetic cataract. In the present study, we used a cDNA microarray method to analyse gene expression patterns in streptozotocin-induced diabetic rats and galactose-fed cataractous lenses. In addition, we investigated the regulation and interaction(s) of anti-oxidant protein 2 and lens epithelium-derived growth factor in these models. METHODS: To identify differential gene expression patterns, one group of Sprague-Dawley rats was made diabetic with streptozotocin and a second group was made galactosaemic. Total RNA was extracted from the lenses of both groups and their controls. Labelled cDNA was hybridised to Atlas Rat Arrays. Changes in gene expression level were analysed. Real-time PCR and western analysis were used to validate the microarray results. RESULTS: The expression of 31 genes was significantly modulated in hyperglycaemic lenses compared with galactosaemic lenses. Notably, transcript and protein levels of B-cell leukaemia/lymphoma protein 2 and nuclear factor-kappaB were significantly elevated in rat lenses at 4 weeks after injection of streptozotocin. At a later stage, mRNA and protein levels of TGF-beta were elevated. However, levels of mRNA for IGF-1, lens epithelium-derived growth factor and anti-oxidant protein 2 were diminished in streptozotocin-induced diabetic cataract. CONCLUSIONS/INTERPRETATIONS: These results provide evidence that progression of sugar cataract involves oxidative- and TGF-beta-mediated signalling. These pathways may promote abnormal gene expression in the hyperglycaemic and galactosaemic states and thus may contribute to the symptoms associated with these conditions. Since oxidative stress seems to be a major event in cataract formation, supply of anti-oxidant may postpone the progression of such disorders.
Assuntos
Catarata/genética , Diabetes Mellitus Experimental/complicações , Galactosemias/complicações , Perfilação da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos , Animais , Apoptose/genética , Catarata/metabolismo , Catarata/patologia , DNA Complementar/genética , Diabetes Mellitus Experimental/induzido quimicamente , Regulação para Baixo/genética , Galactose/farmacologia , Galactosemias/induzido quimicamente , Fator de Crescimento Insulin-Like I/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Cristalino/química , Cristalino/metabolismo , Cristalino/patologia , NF-kappa B/genética , Peroxidases/metabolismo , Peroxirredoxinas , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , Ratos , Ratos Sprague-Dawley , Fator de Crescimento Transformador beta/genética , Regulação para Cima/genética , Proteína X Associada a bcl-2RESUMO
UNLABELLED: Galactosaemia is an inborn error of galactose (Gal) metabolism characterized by irreversible brain damage. The aim of this study was to evaluate whether the antioxidants L-cysteine (Cys) and the reduced glutathione (GSH) could reverse the alterations of brain total antioxidant status (TAS) and the modulated activities of the enzymes Na+,K+ -ATPase and Mg2+ -ATPase in in vitro galactosaemia. Mixture A (mix. A: galactose-1-phosphate (Gal-1-P, 2mM) plus galactitol (Galtol, 2mM) plus Gal (4mM) = classical galactosaemia) or Mixture B (mix. B: Galtol (2mM) plus Gal (1mM) = galactokinase deficiency galactosaemia) were preincubated in the presence or absence of Cys (0.83mM) or GSH (0.83 mM) with whole brain homogenates of suckling rats at 37 degrees C for 1h. TAS and the enzyme activities were determined spectrophotometrically. The preincubation of brain homogenates with mix. A or mix. B resulted in a decrease of TAS to 30% (P < 0.01), while the presence of Cys or GSH increased TAS to 20% (P < 0.01) and 60% ( P < 0.001), respectively. The antioxidants reversed the inhibited Na+,K+ -ATPase by mix. A or mix. B and the stimulated Mg2+ -ATPase by mix. B to control values, whereas no effect was observed on the enormously activated Mg2+ -ATPase by mix. A. CONCLUSIONS: (a) Gal and its derivatives may produce free radicals in the suckling rat brain, reported for first time, (b) Na+,K+ -ATPase inhibition and Mg2+ -ATPase activation are probably due to the oxidative stress from the above compounds, (c) Cys or GSH could play a protective role reversing the inhibited Na+,K+ -ATPase toward normal in in vitro galactosaemia and (d) the addition of the above antioxidants may reduce the consequences of brain Mg2+ -ATPase activation by Gal and Galtol in galactokinase deficiency galactosaemia.
Assuntos
Animais Lactentes/metabolismo , Encefalopatias Metabólicas Congênitas/enzimologia , ATPase de Ca(2+) e Mg(2+)/efeitos dos fármacos , Cisteína/fisiologia , Galactosemias/enzimologia , Glutationa/fisiologia , ATPase Trocadora de Sódio-Potássio/efeitos dos fármacos , Animais , Antioxidantes/química , Antioxidantes/metabolismo , Encéfalo/citologia , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Encefalopatias Metabólicas Congênitas/tratamento farmacológico , Encefalopatias Metabólicas Congênitas/fisiopatologia , ATPase de Ca(2+) e Mg(2+)/antagonistas & inibidores , ATPase de Ca(2+) e Mg(2+)/metabolismo , Cisteína/farmacologia , Combinação de Medicamentos , Avaliação Pré-Clínica de Medicamentos , Feminino , Galactitol/efeitos adversos , Galactitol/química , Galactitol/metabolismo , Galactose/efeitos adversos , Galactose/análogos & derivados , Galactose/metabolismo , Galactosemias/induzido quimicamente , Galactosemias/fisiopatologia , Galactosefosfatos/efeitos adversos , Galactosefosfatos/química , Galactosefosfatos/metabolismo , Glutationa/farmacologia , Masculino , Ratos , Ratos Wistar , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
We demonstrated that aldose reductase inhibition corrects the impaired microvascular responses to inflammatory mediators in diabetic rats. To study the mechanism involved in the restoring effect of aldose reductase inhibition, we examined the effects of minalrestat, another aldose reductase inhibitor, on the responses of mesenteric microvessels studied in vivo to permeability-increasing agents in diabetic and galactosemic rats. The diabetic group was treated from 3 days after the alloxan injection with minalrestat (10 mg/kg/day) for 30 days and the minalrestat treatment (10 mg/kg/day/7 days) of galactosemic rats started concomitantly with the induction of galactosemia. The mesenteric microvessel reactivity was studied using intravital microscopy and changes in vessel diameters were estimated after the topical application of vasoactive agents. The impaired responses to bradykinin, histamine, and platelet-activating factor of arterioles and venules observed in diabetic and galactosemic rats were completely prevented by minalrestat. Neither diabetes nor galactosemia affected responses to acetylcholine and sodium nitroprusside. Responses to these agents were not modified by aldose reductase inhibition. The restoring effect of minalrestat was reversed by inhibition of nitric oxide (NO) synthesis with N(omega)-nitro-L-arginine methyl ester, by blocking K(+) channel with tetraethylammonium but not by cyclooxygenase inhibition with diclofenac. Therefore, we concluded that NO, membrane hyperpolarization, but not cyclooxygenase products are involved in the beneficial effect of minalrestat on the microvascular reactivity in diabetes. Together, these findings led us to suggest that aldose reductase inhibition might ameliorate diabetic complications through the correction of the altered microvascular reactivity by a mechanism that involves NO and membrane hyperpolarization.
Assuntos
Aldeído Redutase/antagonistas & inibidores , Diabetes Mellitus Experimental/fisiopatologia , Imidas/farmacologia , Quinolonas/farmacologia , Circulação Esplâncnica/efeitos dos fármacos , Vasoconstritores/farmacologia , Aldeído Redutase/metabolismo , Animais , Modelos Animais de Doenças , Interações Medicamentosas , Galactosemias/induzido quimicamente , Galactosemias/fisiopatologia , Masculino , Ratos , Ratos WistarRESUMO
PURPOSE: To investigate the effect of termination of galactose feeding after a very short duration of experimental galactosemia on the biochemical abnormalities that are postulated to contribute to the development of retinopathy. METHODS: Experimentally galactosemic rats (normal rats fed a 30% galactose-rich diet for 2 months) were fed a galactose-free diet for an additional 1 month. At the end of 3 months, retinas were removed to measure oxidative stress, nitric oxides (NOs), activity of PKC, and levels of nitrotyrosine. Data were compared between rats in the control group (fed a normal diet) and those in the experimentally galactosemic group (30% galactose diet for the entire 3 months). RESULTS: Interruption of 2 months of galactose feeding by withdrawal of galactose from the diet for 1 additional month had partially beneficial effects on retinal lipid peroxides, but the levels of an endogenous antioxidant, reduced glutathione (GSH), remained subnormal in the retina of galactose-withdrawal rats (P < 0.05 vs. normal and P > 0.05 vs. galactose group). Cessation of the galactose-rich diet had partially beneficial effects on NO levels in the retina, but the levels of nitrotyrosine, an indicator of the formation of peroxynitrite, and activation of PKC were not affected. CONCLUSIONS: The results show that retinal dysmetabolism continues to progress after experimental galactosemia is terminated in rats: Particularly, antioxidant levels remain subnormal, and nitrotyrosine levels are elevated for at least 1 month. Identification of metabolic abnormalities associated with the progression of incipient retinopathy after hyperglycemia is normalized may help in the search for the cause of retinopathy.
Assuntos
Galactosemias/complicações , Doenças Metabólicas/metabolismo , Retina/metabolismo , Doenças Retinianas/metabolismo , Tirosina/análogos & derivados , Animais , Progressão da Doença , Esquema de Medicação , Galactose/administração & dosagem , Galactosemias/induzido quimicamente , Glutationa/metabolismo , Hexoses/sangue , Peróxidos Lipídicos/metabolismo , Masculino , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , Estresse Oxidativo , Proteína Quinase C/metabolismo , Ratos , Ratos Sprague-Dawley , Tirosina/metabolismoRESUMO
PURPOSE: To test the hypothesis that regional retinal oxygenation responses to a hyperoxic inhalation challenge are associated with reported retinopathy outcomes after different therapies in rat models of diabetic retinopathy. METHODS: Six groups of rats were maintained for 3 months: controls (n = 8), untreated diabetic (n = 8), aminoguanidine (AMG)-treated diabetic (2.5 g/kg of diet; n = 6), untreated galactosemic (n = 7), AMG-treated galactosemic (n = 10), and WAY-509-treated (25 mg/kg body weight per day) galactosemic (n = 7). After 3 months, the change in oxygen tension was measured noninvasively from the superior to the inferior ora serrata, using a novel functional magnetic resonance imaging (fMRI) technique and a carbogen (a gas mixture of 5% carbon dioxide and 95% oxygen that has been used clinically, instead of 100% oxygen, to minimize the vasoconstrictive effects of pure O(2) on retinal blood flow and oxygenation) inhalation challenge. Retinal morphometric measurements were also obtained. RESULTS: Retinal lesions (acellular capillaries and pericyte ghosts) were not significantly (P > 0.05) present at 3 months in any experimental groups compared with the control group. Superior but not inferior hemiretinal change in partial pressure of oxygen (DeltaPO(2)) became significantly subnormal (P < 0.05) at 3 months of diabetes or galactosemia. Aminoguanidine, which has been found to inhibit the development of retinopathy in diabetic but not galactosemic rats, inhibited the development of a subnormal DeltaPO(2) in diabetes but not in galactosemia. WAY-509, which has been reported to inhibit retinopathy in galactosemic rats, inhibited the DeltaPO(2) defect in galactosemic rats. CONCLUSIONS: An early subnormal superior hemiretinal DeltaPO(2) after treatment appears to be a good predictor of the risk of development of retinopathy, as well as for assessing therapeutic efficacy in experimental diabetic retinopathy.
Assuntos
Aldeído Redutase/antagonistas & inibidores , Retinopatia Diabética/diagnóstico , Oxigênio/metabolismo , Retina/metabolismo , Aldeído Redutase/uso terapêutico , Animais , Glicemia/metabolismo , Diabetes Mellitus Experimental , Retinopatia Diabética/tratamento farmacológico , Retinopatia Diabética/metabolismo , Inibidores Enzimáticos/uso terapêutico , Galactose/toxicidade , Galactosemias/induzido quimicamente , Galactosemias/metabolismo , Guanidinas/uso terapêutico , Imageamento por Ressonância Magnética , Óxido Nítrico Sintase/antagonistas & inibidores , Consumo de Oxigênio/efeitos dos fármacos , Pressão Parcial , Ratos , Fatores de RiscoRESUMO
Chaperone-like activity and structural changes of lens alpha-crystallin from rats fed with galactose at various time intervals have been studied using high-performance liquid chromatograph (HPLC), circular dichroism (CD), and 1-anilinonaphthalene-8-sulfonic acid (ANS) fluorescence emission. It was found that chaperone-like activity of alpha-crystallin from galactose-fed rats toward dithiothreitol (DTT)-induced insulin B aggregation started to decrease after 3 weeks and decreased significantly after 5 weeks. Consistent results were observed in lens morphology, and lens opacity slightly developed after 3 weeks and became obvious after 5 weeks. HPLC analysis for chaperone function showed that the formation of high molecular weight aggregates (HMWA) of alpha-/gamma-crystallins decreases with the increase of galactose-feeding time, revealing that chaperone-like activity is concomitant with the formation of HMWA. Circular dichroism results showed the reduction of beta-sheet structure and loss of microenvironment of aromatic-type amino acids for opaque lenses, indicating alpha-crystallin's secondary and tertiary structure changed with the development of the lens opacity. ANS binding site estimated by Klotz equation showed it is 1.5 times higher at room temperature and is 2.4 times higher at 58 degrees C for age-matched normal alpha-crystallin than for 5-week galactose-fed lens alpha-crystallin, indicating opaque lens alpha-crystallin loses the ability to assemble into an appropriately placed hydrophobic regions. The overall results accordingly indicated that galactose-induced cataractous alpha-crystallin has disordered structure, leading to the loss of its chaperone-like activity.
Assuntos
Cristalinas/química , Cristalinas/metabolismo , Galactosemias/metabolismo , Galactosemias/patologia , Cristalino/química , Cristalino/patologia , Naftalenossulfonato de Anilina/metabolismo , Animais , Sítios de Ligação , Catarata/induzido quimicamente , Catarata/metabolismo , Catarata/patologia , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Ditiotreitol/farmacologia , Feminino , Galactose/administração & dosagem , Galactose/metabolismo , Galactose/farmacologia , Galactosemias/induzido quimicamente , Insulina/metabolismo , Cristalino/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Peso Molecular , Ligação Proteica/efeitos dos fármacos , Conformação Proteica/efeitos dos fármacos , Desnaturação Proteica , Ratos , Ratos Sprague-Dawley , Espectrometria de Fluorescência , Relação Estrutura-Atividade , Temperatura , Fatores de TempoRESUMO
We attempted to clarify the pattern of cataract development in 12-month-old rats fed a 25% galactose diet and to assess the relation of cataract development with osmotic stress and oxidative damage. In lenses of 12-month-old male Wistar rats fed a 25% galactose diet over an 8-month period, suture accentuation appeared at 6 months of galactose feeding and then opacities developed from the anterior subcapsular cortex toward the posterior subcapsular cortex, reaching the nuclear region at 8 months of galactose feeding. Increases in lens galactitol and lipid peroxide contents and a decrease in lens reduced glutathione content occurred at 4, 6 and 8 months of galactose feeding. The increase in lens lipid peroxide content and the decrease in lens reduced glutathione content were accelerated with an increase in feeding period, while the increase in lens galactitol content was decelerated. An increase in lens water content and a decrease in lens protein content occurred at 6 and 8 months of galactose feeding. The lens vitamin E content increased at 6 months of galactose feeding and this increase was concomitant with increases in serum vitamin E and total cholesterol concentrations. The serum lipid peroxide concentration increased at 4 and 6 months of galactose feeding. The present results indicate that in lenses of 12-month-old rats fed a 25% galactose diet, suture accentuation appears initially and then opacities develop from the anterior subcapsular cortex toward the posterior subcapsular cortex, finally reaching the nuclear region. These results also suggest that in the galactosemic aged rats, osmotic stress would mainly contribute to cataract formation, while oxidative damage could be linked to both cataract formation and progression, although an increase in lens vitamin E content occurs during the cataract development.
Assuntos
Água Corporal/metabolismo , Catarata/induzido quimicamente , Galactose/efeitos adversos , Cristalino/efeitos dos fármacos , Estresse Oxidativo , Animais , Catarata/metabolismo , Catarata/patologia , Colesterol/sangue , Cristalinas/metabolismo , Dieta , Galactitol/metabolismo , Galactosemias/induzido quimicamente , Galactosemias/metabolismo , Galactosemias/patologia , Glutationa/metabolismo , Cristalino/metabolismo , Cristalino/patologia , Peróxidos Lipídicos/sangue , Masculino , Pressão Osmótica , Ratos , Ratos Wistar , Vitamina E/sangueAssuntos
Aldeído Redutase/antagonistas & inibidores , Retinopatia Diabética/prevenção & controle , Fatores de Crescimento Endotelial/biossíntese , Inibidores Enzimáticos/farmacologia , Galactosemias/metabolismo , Guanidinas/farmacologia , Linfocinas/biossíntese , Animais , Retinopatia Diabética/metabolismo , Galactose , Galactosemias/induzido quimicamente , Hemoglobinas Glicadas/metabolismo , Ratos , Retina/metabolismo , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
In the retinas of diabetic animals, protein kinase C (PKC) activity is elevated, and Na+-K+-ATPase and calcium ATPase activities are subnormal. These abnormalities are also present in another model of diabetic retinopathy, experimental galactosemia. We have investigated the relationship between hyperglycemia-induced abnormalities of PKC and ATPases using a selective inhibitor of beta isoform of PKC (LY333531). Diabetes or experimental galactosemia of 2 months' duration resulted in > 50% elevation of PKC activity in the retina, and administration of LY333531 prevented the elevation. In retinas of the same rats, the LY333531 prevented hyperglycemia-induced decreases of both Na+-K+-ATPase and calcium ATPase activities. Retinal microvessels, the main site of lesions in diabetic retinopathy, likewise showed elevated activity of PKC and inhibition of ATPases in diabetes and in experimental galactosemia, and administration of LY333531 to diabetic animals prevented these abnormalities. PKC activity in sciatic nerves, in contrast, became subnormal in diabetes and experimental galactosemia, and LY333531 had no effect on PKC activity in the sciatic nerve. PKC activity in the cerebral cortex was not affected by diabetes or experimental galactosemia. The results suggest that diabetes-induced reductions in Na+-K+-ATPase and calcium ATPase in the retina are mediated in large part by PKC-beta. The availability of an agent that can normalize the hyperglycemia-induced increase in PKC activity in the retina should facilitate investigation of the role of PKC in the development of diabetic retinopathy.
Assuntos
Diabetes Mellitus Experimental/enzimologia , Galactosemias/enzimologia , Proteína Quinase C/metabolismo , Retina/enzimologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/enzimologia , Estudos de Coortes , Inibidores Enzimáticos/sangue , Inibidores Enzimáticos/farmacologia , Galactosemias/induzido quimicamente , Indóis/sangue , Indóis/farmacologia , Maleimidas/sangue , Maleimidas/farmacologia , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/efeitos dos fármacos , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Retina/efeitos dos fármacos , Nervo Isquiático/efeitos dos fármacos , Nervo Isquiático/enzimologia , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , ATPase Trocadora de Sódio-Potássio/efeitos dos fármacosRESUMO
PURPOSE: The purpose of the study was to examine the effect of topical application of substance P (SP), insulin-like growth factor-1 (IGF-1) and vasoactive intestinal polypeptide (VIP) on corneal re-epithelialization in galactosemic rats. METHODS: Experimental galactosemia was induced by feeding a diet containing 30% galactose for 4-6 months. The corneal epithelium was debrided bi-laterally by scraping with a blunted scalpel blade. One eye (control) received only a saline solution whilst the other eye received a solution of SP and/or IGF-1 or VIP. A single drop of control or test solution was administered 4 times daily until wound closure. Corneas were stained with fluorescein and videotaped under ultraviolet illumination at regular time intervals after debridement. After digitizing the video image, the wound area was calculated using an image analysis program (NIH Image). RESULTS: Corneal re-epithelialization was significantly delayed in galactosemic compared to normal animals. Rates of healing were 1.3 +/- 0.07 mm2/hour and 1.02 +/- 0.02 mm2/hour for normal and galactosemic animals, respectively. Topical application of SP in concentrations ranging from 25 pg/ml up to 250 microg/ml had no significant effect on the rate of corneal re-epithelialization. Similarly, IGF-1 (1 microg/ml) or VIP (1 microg/ml) when applied alone did not affect re-epithelialization. Furthermore, resurfacing of the debrided area was not affected by co-application of SP (250 microg/ml) and IGF-1 or VIP. CONCLUSIONS: Independent or combined topical application of SP, VIP or IGF-1 at the concentrations tested, does not modulate corneal epithelial wound healing in rats with galactosemia induced by 30% galactose.
Assuntos
Córnea/fisiologia , Galactosemias/fisiopatologia , Fator de Crescimento Insulin-Like I/farmacologia , Regeneração/fisiologia , Substância P/farmacologia , Peptídeo Intestinal Vasoativo/farmacologia , Cicatrização/fisiologia , Animais , Córnea/citologia , Córnea/efeitos dos fármacos , Desbridamento , Quimioterapia Combinada , Epitélio/lesões , Epitélio/fisiologia , Galactose , Galactosemias/induzido quimicamente , Processamento de Imagem Assistida por Computador , Fator de Crescimento Insulin-Like I/administração & dosagem , Masculino , Soluções Oftálmicas , Ratos , Ratos Sprague-Dawley , Regeneração/efeitos dos fármacos , Substância P/administração & dosagem , Peptídeo Intestinal Vasoativo/administração & dosagem , Cicatrização/efeitos dos fármacosRESUMO
OBJECTIVE: To study the effects of an aldose reductase inhibitor (ARI-509, Wyeth-Ayerst, Princeton, NJ) and aminoguanidine (AMG), agents that have been reported to prevent or delay diabetic retinopathy, on retinal vascular abnormalities and the immunocytochemical expression in the retina of vascular endothelial growth factor (VEGF) in rats maintained for up to 2 years on a 50% galactose diet. METHODS: Albino rats were placed on a control diet, a diet containing 50% galactose, or the 50% galactose diet containing either ARI-509 or AMG. Treatment with ARI-509 or AMG was initiated at the beginning of the experiment or after 12 months of galactose feeding. After 22 to 24 months, the rats were killed and the retinal vasculature from half of one eye was isolated by trypsin-elastase digestion for semiquantitative evaluation of retinal vascular lesions. The other half of the retina was prepared for immunocytochemistry and stained for the presence of VEGF, factor VIII, vimentin, and glial fibrillary acidic protein. Red blood cells, sciatic nerves, and a portion of the retina from the second eye were assayed for glucose, galactose, fructose, sorbitol, galactitol, and myo-inositol. Red blood cells were also assayed for galactosylated hemoglobin. RESULTS: Galactose-fed animals developed a vascular retinopathy characterized by severe cellular loss in the retinal capillaries and intensification of periodic acid-Schiff staining of the vascular basement membranes. Some animals also displayed dilation and hypercellularity of vessels in the posterior retina. These changes were substantially reduced in animals receiving ARI-509 from the beginning of the galactose diet, but were unaffected in all of the other treatment groups. None of the rats receiving ARI-509 or AMG treatment, whether initiated from the onset or after 12 months of galactosemia, demonstrated VEGF immunoreactivity. With the exception of the animals receiving ARI-509 from the beginning of the experiment, all of the galactose-fed animals developed dense cataracts within 6 weeks of the beginning of the galactose diet. Galactitol levels in animals receiving ARI-509 were 86% to 93% lower in red blood cells, retina, and sciatic nerve than those in the other galactose-fed groups. CONCLUSIONS: Although ARI-509 and AMG have different abilities to delay or prevent the diabetic-like retinopathy in galactosemic rats, even when substantial retinal microvascular acellularity occurs, both drugs prevent the immunocytochemical expression of VEGF. These results suggest that factors other than hypoxia may be responsible for VEGF expression in the retina, and that aldose reductase inhibitors and AMG have potential roles in preventing such expression and, thus, perhaps preventing retinal neovascularization.
Assuntos
Aldeído Redutase/antagonistas & inibidores , Fatores de Crescimento Endotelial/biossíntese , Inibidores Enzimáticos/farmacologia , Galactosemias/metabolismo , Guanidinas/farmacologia , Linfocinas/biossíntese , Retina/efeitos dos fármacos , Aldeído Redutase/farmacologia , Animais , Catarata/induzido quimicamente , Catarata/patologia , Retinopatia Diabética/metabolismo , Retinopatia Diabética/patologia , Retinopatia Diabética/prevenção & controle , Fator VIII/metabolismo , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Galactose , Galactosemias/induzido quimicamente , Galactosemias/patologia , Proteína Glial Fibrilar Ácida/metabolismo , Ratos , Ratos Sprague-Dawley , Retina/metabolismo , Retina/patologia , Vasos Retinianos/efeitos dos fármacos , Vasos Retinianos/metabolismo , Vasos Retinianos/patologia , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio Vascular , Vimentina/metabolismoRESUMO
PURPOSE: Magnetic resonance imaging (MRI) is becoming increasingly important for the diagnosis and characterization of ocular pathologies. A drawback to this technique is that image contrast between different regions of tissue can be obscured because of the similarity of their nuclear magnetic resonance relaxation parameters. This problem is addressed by magnetization transfer contrast (MTC) enhancement, a MRI technique that generates high-contrast images based on characteristic tissue differences resulting from the interaction of water and macromolecules. The purpose of this study was to investigate the feasibility of using MTC-enhanced imaging to monitor quantitatively the lens changes associated with sugar cataract formation in galactose-fed dogs. METHODS: Male beagles fed a diet containing 30% galactose were periodically examined by MRI for changes in tissue character. Each examination included a gradient recalled echo image (M0), an MTC-enhanced gradient recalled echo image (Ms), a T1 image determined from a one-shot T1 imaging sequence, and a T1-weighted image taken from the raw T1 data. Average values were obtained for several regions of interest and tabulated. These were correlated with cataractous stages visually observed by slit lamp biomicroscopy and retroillumination photography. RESULTS: Enhanced image details of the lens and anterior segment that documented osmotic changes from initial cortical vacuole formation to cortical and nuclear changes associated with advanced sugar cataracts were characterized from measurements of parameters obtained from M0, Ms, T1-weighted, and T1 images. Changes in the cross-sectional areas of lenses during sugar cataract formation also were documented. The magnetic resonance images showed visible changes from the onset of cortical vacuole formation. Region of interest (ROI) analysis of the images showed tissue changes occurring throughout the cataract progression. CONCLUSIONS: The MTC-enhanced MRI technique is well suited to detecting lens changes associated with cataractogenesis. All but the earliest changes were readily apparent from the images with no further analysis. Graphic ROI analysis was able to detect regional changes associated the cataract progression for all degrees of severity. Furthermore, the images demonstrated changes in size and shape that would not be detectable by visual inspection.
Assuntos
Catarata/patologia , Galactosemias/patologia , Cristalino/patologia , Imageamento por Ressonância Magnética/métodos , Animais , Catarata/induzido quimicamente , Catarata/fisiopatologia , Progressão da Doença , Cães , Galactose , Galactosemias/induzido quimicamente , Galactosemias/fisiopatologia , Cristalino/fisiopatologia , Masculino , Índice de Gravidade de DoençaAssuntos
Diabetes Mellitus Experimental/patologia , Retinopatia Diabética/patologia , Galactosemias/patologia , Animais , Diabetes Mellitus Experimental/induzido quimicamente , Retinopatia Diabética/induzido quimicamente , Modelos Animais de Doenças , Galactose , Galactosemias/induzido quimicamente , Ratos , Retina/patologiaRESUMO
The lens epithelial cells are a primary site of involvement in galactosemia. Changes in their size, shape and proliferative capacity have been observed upon exposure to high galactose. In this report, changes in the cell cycle pattern of normal and galactosemic lens epithelial cells were examined by use of flow cytometry. Both changes in DNA and RNA were observed using the fluorochrome, acridine orange. Under the appropriate conditions acridine orange can be used to differentiate double-stranded DNA from single-stranded RNA. Using this approach, the DNA and RNA of normal and galactosemic (1, 4, or 7 days) lens epithelial cells can be compared. The results indicate that lens epithelial cells, when exposed to 40 mM galactose media or 30 mM glucose for 7 days, are induced to enter mitosis. Mannitol did not mimic these results. Changes in the cell cycle pattern were not observed when the cells were treated for 1 or 4 days. Although higher numbers of cells in mitosis were observed after 7 days exposure to 40 mM galactose, a correlation between proliferation, as measured by 3H-thymidine uptake, and mitosis was not possible. Apoptosis was evaluated as a possible explanation for these results. The changes in the DNA staining pattern could be use to monitor lens epithelial cells during galactosemia.
Assuntos
Laranja de Acridina , DNA/análise , Galactosemias/metabolismo , Cristalino/química , RNA/análise , Animais , Bovinos , Ciclo Celular/efeitos dos fármacos , Células Cultivadas , DNA/biossíntese , Epitélio/química , Epitélio/efeitos dos fármacos , Citometria de Fluxo/métodos , Galactose , Galactosemias/induzido quimicamente , Glucose/farmacologia , Cristalino/citologia , Cristalino/efeitos dos fármacos , Mitose/efeitos dos fármacos , Coloração e Rotulagem/métodosRESUMO
Specular microscopic studies indicate that the size (polymegathism) and shape (pleomorphism) of the hexagonal corneal endothelial cells change in diabetics. Similar morphometric changes of the corneal endothelium have also been experimentally observed in diabetic rats as well as in diabetic and galactose-fed dogs and concomitant administration of aldose reductase inhibitors reduced these morphological changes. The purpose of this study was to examine whether corneal endothelial changes in galactose-fed dogs are reversible by the marked reduction of galactitol production after stopping prolonged galactose feeding. Ten control dogs were fed a normal diet, while 48 dogs were fed a diet containing 30% galactose. The galactose diet was removed from 15 dogs after 24 months at which time pericyte ghosts in the retina had developed and another 15 dogs were removed from the galactose diet after 31 months when retinal microaneurysms had developed. Eighteen dogs remained on galactose diet throughout the study (38 months). Specular microscopy was conducted on members of all groups after 38 months of study and the photographs were analyzed in masked fashion on the Bambi image analysis system. The evaluation of the corneal endothelial cells revealed significant differences in the cell size and density between galactose-fed dogs in the three groups and normal, age-matched control dogs. Corneal endothelial changes were not significantly reduced in dogs fed galactose for either 24 months or 31 months and then fed a normal diet for 14 and 7 months, respectively, indicating that amelioration of endothelial cell changes requires therapy prior to the advent of endothelial morphologic changes.
Assuntos
Endotélio Corneano/patologia , Galactose/administração & dosagem , Galactosemias/patologia , Animais , Contagem de Células/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Dieta , Cães , Endotélio Corneano/efeitos dos fármacos , Endotélio Corneano/metabolismo , Galactitol/metabolismo , Galactosemias/induzido quimicamente , MasculinoAssuntos
Retinopatia Diabética/patologia , Galactosemias/patologia , Animais , Retinopatia Diabética/induzido quimicamente , Retinopatia Diabética/metabolismo , Modelos Animais de Doenças , Cães , Galactose , Galactosemias/induzido quimicamente , Galactosemias/metabolismo , Disco Óptico/irrigação sanguínea , Neovascularização Retiniana/induzido quimicamente , Neovascularização Retiniana/patologia , Sorbitol/metabolismoRESUMO
BACKGROUND: Progression of diabetic retinopathy in human subjects and animal models is difficult to halt promptly by intensified insulin therapy and strict glycemic control. OBJECTIVE: To learn whether this resistance to arrest is peculiar to diabetes and insulin therapy or is a characteristic of hyperglycemia itself, we have determined the effect of intervention on diabetic-like retinopathy in a non-diabetic animal model, the galactose-fed dog. METHODS: Dogs were given a 30% galactose diet. At the end of 24 months, the dogs were divided into two groups, one of which continued to receive the galactose diet, while the second immediately began receiving the diet minus galactose. All animals were killed after 60 months of study. RESULTS: Consumption of the galactose-rich diet resulted, as expected, in galactosemia evident by elevated hemoglobin A1, plasma nonenzymatically glycated protein, and erythrocyte polyol concentrations, each of which decreased to normal levels following withdrawal of dietary galactose. Retinopathy was found to be equivocal at the end of 24 months of the galactose diet and subsequently progressed significantly despite cessation of the galactose diet. CONCLUSION: The fact that retinopathy did not halt promptly at intervention is consistent with previous data from diabetic dogs and suggests that the vascular lesions may be due to sequelae of excessive tissue aldohexose that are not promptly corrected by correction of the aldohexose level.
Assuntos
Retinopatia Diabética/fisiopatologia , Galactosemias/fisiopatologia , Vasos Retinianos/patologia , Animais , Análise Química do Sangue , Diabetes Mellitus Experimental/fisiopatologia , Retinopatia Diabética/induzido quimicamente , Retinopatia Diabética/patologia , Modelos Animais de Doenças , Progressão da Doença , Cães , Galactose , Galactosemias/induzido quimicamente , Hiperglicemia/fisiopatologiaRESUMO
Galactose-fed rats develop a retinal microvascular disease, but retinopathy has not been found to develop reproducibly in diabetic rats. We sought to determine which retinal lesions can be reproducibly produced by long-term diabetes in rats, the extent to which the capillary lesions in diabetic rats and galactosemic rats are similar, and whether the retinopathy induced by 50% galactose can be reproduced satisfactorily by a lower concentration of galactose. Alloxan-diabetic rats and rats fed either a 50% galactose diet or a 30% galactose diet were killed after comparable durations of study (18 to 22 months). Rats fed 50% galactose showed greater than normal frequency of retinal pericyte ghosts and acellular capillaries, and thickening of capillary basement membranes by 18 months of galactosemia. Rats eating 30% galactose developed similar retinal lesions, and tended to be healthier than rats fed 50% galactose. Diabetes of 1 1/4 years or more likewise resulted in retinal pericyte ghosts, acellular capillaries and thickened capillary basement membrane. IRMAs and other vascular abnormalities were not reproducibly demonstrated at this duration of study, and saccular microaneurysms were not seen in any groups. In a number of diabetic rats, the severity of diabetes diminished spontaneously (after 1 to 1 1/2 years of insulin deficiency), thus making it essential that glycemia be systematically monitored. Both diabetic rats and experimentally galactosemic rats develop microvascular lesions that are consistent with at least the early stages of diabetic retinopathy, and these models should be useful to screen potential therapies for their ability to inhibit the development of retinopathy.
Assuntos
Diabetes Mellitus Experimental/patologia , Retinopatia Diabética/patologia , Galactosemias/patologia , Vasos Retinianos/patologia , Aloxano , Animais , Membrana Basal/patologia , Capilares/patologia , Diabetes Mellitus Experimental/induzido quimicamente , Retinopatia Diabética/induzido quimicamente , Galactose , Galactosemias/induzido quimicamente , Hemoglobinas Glicadas/análise , Hiperglicemia/induzido quimicamente , Hiperglicemia/patologia , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Levels of the intracellular antioxidant, glutathione, become subnormal in retina in diabetes or experimental galactosemia. In order to investigate the cause and significance of this abnormality, activity of gamma-glutamyl transpeptidase (an enzyme important in the synthesis and degradation of glutathione) and levels of reduced glutathione have been measured in retinas of diabetic rats and dogs and of experimentally galactosemic rats and dogs. Retinal gamma-glutamyl transpeptidase activity and glutathione level were significantly less than normal after 2 months of diabetes or galactosemia. In contrast, cerebral cortex from the same diabetic rats and galactosemic rats showed no significant reduction in either gamma-glutamyl transpeptidase activity or glutathione level. These different responses of the two tissues to hyperglycemia might help account for the difference in microvascular disease in these two tissues in diabetes. Consumption of the antioxidants, ascorbic acid (1.0%) plus alpha-tocopherol (0.1%), by diabetic rats and galactosemic rats inhibited the decrease of gamma-glutamyl transpeptidase activity and glutathione levels in retina, suggesting that defects in glutathione regulation in the retina are secondary to hyperglycemia-induced 'oxidative stress'.