This paper outlines the progression of age-related macular degeneration in the eye and discusses the diagnostic approaches and therapies used currently to treat this disease. Age-related macular degeneration has a complicated pathophysiology involving genetic and environmental factors. This paper focuses its attention on the role of lipofuscin accumulation in this disease. Lipofuscin in the eye refers to the bisretinoid products of the visual cycle. While lipofuscin accumulation is normal in healthy eyes, the excessive accumulation causes retinal dysfunction. Lipofuscin accumulation has been linked heavily not only to age-related macular degeneration but also juvenile macular degeneration, retinitis pigmentosa, Best’s Villiform disease, and many others. New techniques in ophthalmic research have evaluated the role of lipofuscin accumulation in such retinal genetic diseases. This paper proposes an approach to apply techniques such as quantified autofluorescence imaging and high-powered liquid chromatography of bisretinoids in the eye to track the role of lipofuscin accumulation in the progression of age-related macular degeneration.

Diss. (sammanfattning) Linköping : Univ., 2002. / Härtill 4 uppsatser.

by Hiu-ming Li. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1993. / Includes bibliographical references (leaves 81-88). / SUMMARY --- p.1 / Chapter CHAPTER 1. --- INTRODUCTION --- p.3 / Chapter CHAPTER 2. --- LITERATURE REVIEW --- p.7 / Chapter 2.1. --- Retinal pigment epithelium --- p.7 / Chapter 2.1.1. --- Embryology / Chapter 2.1.2. --- Anatomy and histology / Chapter 2.1.3. --- Growth and aging / Chapter 2.1.4. --- Macular region / Chapter 2.2. --- The photoreceptor outer segment --- p.12 / Chapter 2.3. --- Lipofuscin --- p.13 / Chapter 2.4. --- Lipofuscin in retinal pigment epithelium and retinal photoreceptor disc shedding --- p.14 / Chapter 2.5. --- Possible mechanism for lipofuscin formation in the RPE --- p.22 / Chapter 2.6. --- Age-related lipofuscin accumulation in the RPE --- p.22 / Chapter 2.7. --- Racial difference of RPE lipofuscin concentration --- p.25 / Chapter 2.8. --- RPE lipofuscin and age-related macular degeneration --- p.26 / Chapter CHAPTER 3. --- MATERIALS AND METHODS --- p.28 / Chapter 3.1. --- Histologic Specimens --- p.28 / Chapter 3.2. --- Measuring equipment --- p.28 / Chapter 3.3. --- Software of measurements --- p.31 / Chapter 3.4. --- Light source and filters --- p.31 / Chapter 3.5. --- Control --- p.31 / Chapter 3.6. --- Measurement of autofluorescent Intensity --- p.32 / Chapter 3.7. --- Bleaching (oxidation) of melanin --- p.39 / Chapter CHAPTER 4. --- RESULTS --- p.42 / Chapter 4.1. --- Bleaching test --- p.42 / Chapter 4.2. --- RPE autofluorescence observation in different age --- p.43 / Chapter 4.3. --- RPE autofluorescence observation within individual eyes --- p.45 / Chapter 4.4. --- Topographic distribution of lipofuscin --- p.49 / Chapter 4.5. --- Lipofuscin content at the Foveola --- p.50 / Chapter 4.6. --- The relationship of age and lipofuscin content in total RPE --- p.51 / Chapter 4.7. --- The relationship of age and lipofuscin content in the macular RPE --- p.57 / Chapter 4.8. --- Relationship of age and lipofuscin content in the posterior pole of RPE --- p.59 / Chapter 4.9. --- Relationship of age and lipofuscin content in the temporal RPE --- p.61 / Chapter 4.10. --- Relationship of age and lipofuscin content in the nasal RPE --- p.63 / Chapter 4.11. --- Age related topographic changes --- p.65 / Chapter 4.12. --- The relationship of age and lipofuscin content in the RPE of male --- p.66 / Chapter 4.13. --- The relationship of age and lipofuscin content in the RPE of female --- p.68 / Chapter 4.14. --- Relationship of lipofuscin content in different sex --- p.70 / Chapter CHAPTER 5. --- DISCUSSION --- p.71 / Chapter 5.1. --- Evaluation of method --- p.71 / Chapter 5.2. --- RPE lipofuscin content in different age --- p.71 / Chapter 5.3. --- Topographic distribution of lipofuscin --- p.74 / Chapter 5.4. --- Lipofuscin and age-related macular degeneration in Chinese --- p.78 / REFERENCES --- p.81

Microglia are a unique type of immune cell found within the brain, spinal cord and retina. In the healthy brain, their job is to support neurons, defend against infectious microbes, clear extracellular debris and remove dead or dying cells through phagocytosis. This diverse array of functions presents the possibility of unique subsets of microglia existing in the healthy brain, yet none have been described thus far. By utilizing cellular autofluorescence as a discriminating characteristic, we identified two novel subsets of microglia present in the healthy brains of mice and non-human primates. Approximately 70% of microglia displayed autofluorescence (AF+) while the remaining 30% did not (AF–). While the proportion of AF+ and AF– microglia remained constant throughout most of adult life, the autofluorescence intensity increased exclusively in the AF+ subset at an almost linear rate with age. This gain in autofluorescence correlated with equivalent increases in the size and complexity of storage bodies, as detected by transmission electron microscopy and increases in LAMP1 levels, a key component of the lysosomal compartment. As the brain ages, lysosomal storage material builds up inside AF+ microglia, further increasing the accumulation of autofluorescence as a result. The analysis of protein content in autofluorescent subsets revealed that AF+ microglia produced more proteins and enzymes involved in the storage and degradation of waste material, as well as more proteins involved in the regulation of mTOR, a key cellular pathway governing nutrient availability and energy production. Interestingly, the disruption of lysosomal function in microglia through genetic mutations accelerated the accumulation of storage material in AF+ cells, which led to impaired microglia physiology and increased cell death, mimicking the effects observed during advanced aging. Increasing evidence suggests that the accumulation of waste materials inside the brain contributes to diseases of aging and these data are suggestive of a mechanistic convergence between aging and lysosomal storage disorders.

The lysosomal compartment is a major site for intracellular degradation. Lysosomal degradation of the cell’s own constituents, so-called autophagy, not only provides a cell with nutrients, but also removes damaged and potentially dangerous endogenous structures, thus securing intracellular homeostasis. On the other hand, lysosomes have been shown to be involved in the initial stages of apoptosis, and the protective effect of autophagy has been suggested to switch to cell death when excessive. Ageing-related changes of cellular structures result from damage caused by eactive oxygen species (ROS), which are an inevitable by-product of aerobic life. Intracellular turnover of compromised organelles and macromolecules, to which lysosomal degradation is a major contributor, does not function perfectly, even under favourable conditions. This inherent incompleteness of lysosomal degradation is responsible for the accumulation of a variety of nondegraded and functionally inefficient structures, which can be considered biological “garbage”. Biological “garbage” includes damaged non-degraded macromolecules and organelles, as well as intralysosomal non-degradable polymer-like structure called lipofuscin, or age pigment. Although accumulation of biological “garbage” has been suggested harmful, little is known about the mechanisms of its deleterious effects. To gain a better understanding of ageing-related changes of the lysosomal compartment and their influence on cell functions, we focused on studying: (1) the role of macroautophagy in the turnover of organelles and lipofuscin formation; (2) the role of biological “garbage” accumulation in the development of ageing-related changes and eventual death of growth-arrested, postmitotic-like cells; (3) the possible cell-protective effect of mitosis; (4) the influence of lipofuscin on cell survival during complete starvation; and (5) the effects of lipofuscin on lysosomal stability. As a model of induced biological “garbage” accumulation we used confluent human fibroblasts treated with the autophagy inhibitor 3-methyladenine (3MA). Alternatively, lysosomal degradation was suppressed by using the cysteine protease inhibitor leupeptin, or the cathepsin D inhibitor pepstatin A. As a cellular model of aged cells, we used lipofucsin-loaded human fibroblasts. Lipofuscin-loading was achieved by culturing confluent fibroblasts under hyperoxic conditions for 2-4 months. Using these in vitro models, the present study shows that: (1) inhibition of autophagy results in accumulation of lysosome-associated autofluorescent material and mitochondria with low membrane potential; (2) detrimental effect of biological “garbage” accumulation following inhibition of autophagy is prevented by continuous cell division; (3) lipofuscin-loaded cells are more resistant to starvation-induced cell death than control cells; (4) lysosomes of lipofuscinloaded fibroblasts are more resistant to the organelle-targeted stress then lysosomes of control cells. Based on the results of the present study we conclude that properly operating autophagic machinery plays a crucial role in preventing age-related changes associated with accumulation of biological “garbage”. We also suggest that continual proliferation is the natural mechanism by which cells cope with the accumulation of non-degradable material, employing mechanical dilution during the cell division. Finally, we introduce an idea of lipofuscin being a hormetic agent, and possibly possessing some lysosome-stabilising properties. Better understanding of the influence of the age-related accumulation of biological “garbage” on cellular functions may be helpful for future development of anti-ageing therapy and management of age-associated pathologies.

The blue crab, Callinectes sapidus, represents an ecologically and economically important component of marine and estuarine ecosystems. In Florida, blue crab landings accounted for $9.6 million dollars during the 2010 fishing season. Accurate stock assessments for this valuable fishery are essential. Age is a critical biological component of accurate stock assessments; however, blue crabs and other crustaceans are especially difficult to age because of the complex nature of discrete growth. Biochemical extraction of an aging pigment, lipofuscin, was developed using blue crab eyestalks. The current study investigated the effects of freezing preservation on lipofuscin extracts and examined whether the extraction methodology, developed by Chesapeake Bay researchers, was useful for aging Tampa Bay blue crabs populations. Significant differences in lipofuscin index were found between samples frozen (2 weeks at -80°C) prior to analysis and those processed and assayed immediately (p < 0.001). Quarterly assays of the cohort of known-age individuals revealed a negative linear trend (y = -0.12x + 0.49, p < 0.001) in lipofuscin index over a 12-month period. This result suggests that extraction of lipofuscin is not appropriate for age determination of Florida blue crabs. Investigations into possible causes of the negative trend in lipofuscin suggest this method deserves further examination and refinement before it is acceptable as a reliable method for age determination in Florida blue crabs. Growth data of the known-age population collected during this study revealed that blue crabs in Tampa Bay can reach exploitable size in under sixth months and female crabs can reach sexual maturity within seven months of hatching. These growth patterns have the potential to enhance future Florida stock assessments.

No description available.

In this work, the detailed analysis of GABAB-R/G protein coupling in the course of pre- and postnatal development of rat brain cortex indicated the significant intrinsic efficacy of GABAB-receptors already shortly after the birth: at postnatal day 1 and 2. Subsequently, both baclofen and SKF97541-stimulated G protein activity, measured as the high-affinity [35 S]GTPγS binding, was increased. The highest level of agonist-stimulated [35 S]GTPγS binding was detected at postnatal days 14 and 15. In older rats, the efficacy, i.e. the maximum response of baclofen- and SKF97541-stimulated [35 S]GTPγS binding was continuously decreased so, that the level in adult, 90-days old rats was not different from that in newborn animals. The potency of G protein response to baclofen stimulation, characterized by EC50 values, was also high at birth but unchanged by further development. The individual variance among the agonists was observed in this respect, as the potency of SKF97541 response was decreased when compared in 2-days old and adult rats. The highest plasma membrane density of GABAB-R, determined by saturation binding assay with specific antagonist [3 H]CGP54626AA, was observed in 1-day old animals. The further development was reflected in decrease of receptor number. The adult level was ≈3- fold lower than...

Free radicals are highly reactive species with one unpaired electron in orbital. Reactive oxygen and nitrogen species count among important biologic molecules of radical nature. It is very important to fix a concentration of free radicals in cell on non-toxic limits. Products of radical damage are cumulated extra or intracellulary and they are main components of lipofuscin-like pigments. Lipofuscin-like pigments contains in their molecular structure fluorofores, so they are good substrates for fluorescent analysis. Alzheimer's disease is a very actual social and economical problem. Etiology of Alzheimer's disease is still unknown. Histologically, the characteristic presence of Alzheimer's disease is a senil plaques of amyloide ?. ROS and RNS diffuse through hematoencephalic barrier in vessel's lumen and attacks red blood cells. Radical damage of erythrocytes is associated with an increase of concentration of oxidative stress products in cytosol. Sample for fluorescent analysis has been prepared from a red-blood cell extract from 30 patients and 8 healthy controls. Fluorescent spectra of healthy controls have emission maxima in area 327-343nm. In compare with controls, spectra of pacients were more heterogenous in area upon 380nm. The study of oxidative cell damage is important for understanding of...

Orientador: Humberto Santo Neto / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Médicas / Made available in DSpace on 2018-08-13T22:18:59Z (GMT). No. of bitstreams: 1 Oggiam_DaniellaSilva_M.pdf: 1790150 bytes, checksum: f55f529364fdf6fa2bae8c646e2b2d8b (MD5) Previous issue date: 2009 / Resumo: A distrofia Muscular de Duchenne (DMD) é uma miopatia progressiva causada por uma doença autossômica recessiva ligada ao sexo, que acomete crianças do sexo masculino, e evolui para incapacidade motora na puberdade até causar óbito ao redor da segunda década de vida. É causada por uma alteração no gene codificador da proteína distrofina, que mantém a integridade do sarcolema da fibra muscular. O camundongo mdx é utilizado como modelo experimental da DMD para investigações do tecido muscular esquelético e cardíaco, por apresentar muitas semelhanças com humano portador da doença. Na DMD os pacientes iam á óbito por falência respiratória, desde a evolução do tratamento com técnicas de ventilação mecânica artificial, as disfunções cardíacas tornaram-se importantes, visto que a partir disto, a maioria dos óbitos começaram a ser em função da falência cardíaca, que resulta de um processo seguido de: necrose, inflamação, fibrose evoluindo para cardiomiopatia grave. Várias estratégicas farmacológicas tem sido utilizadas para melhora da função cardíaca tanto no portador de DMD como no camundongo mdx. Um dos medicamentos utilizados é o deflazacorte, um glucocorticóide de ação anti-inflamatória, administrado por toda vida do portador de DMD. Embora os efeitos do deflazacorte sobre a função cardíaca no humano e no camundongo mdx têm sido extensivamente estudados, pouco se sabe sobre os efeitos histopatológicos no tecido cardíaco. Neste trabalho foi avaliado o efeito da administração a longo prazo de deflazacorte na progressão da fibrose miocárdica intersticial em camundongos mdx de 6 meses de idade. Os animais foram tratados diariamente com deflazacorte durante 15 meses, após foram sacrificados, o coração foi removido e congelado em nitrogênio líquido para posterior análise histológica e morfológica do tecido. O coração do grupo de camundongos mdx tratados com deflazacorte foi comparado com camundongos mdx não tratados. As áreas de fibrose miocárdica diminuíram significativamente 40% em relação ao grupo não tratado. Concluiu-se que o tratamento à longo prazo com deflazacorte é eficiente para diminuir a progressão da fibrose cardíaca. Sendo assim, como a cardiomiopatia está diretamente relacionada à disfunções celulares que acarreta a necrose dos cardiomiócitos, é de interesse investigar o acúmulo de lipofuscina, um biomarcador do envelhecimento, nos corações de camundongos mdx. Neste trabalho também foi observado o acúmulo de lipofuscina em animais controle C57BL10 e mdx de 14 dias a 23 meses de idade sem serem submetidos a qualquer tratamento. Os animais foram sacrificados, o coração removido e congelado em nitrogênio líquido para posterior análise da fluorescência dos grânulos de lipofuscina. Após contagem dos grânulos observou-se que aumentam com a idade, e dos 4 para os 6 meses ocorreu um acréscimo no acúmulo de lipofuscina. Considerando-se que o acúmulo de lipofuscina relaciona-se a disfunção celular é possível que isto contribua para lesão de cardiomiócitos em corações desprovidos de distrofina / Abstract: The Duchenne Muscle Dystrophy is a progressive myopathy caused by recessive autossomic disease connected to the gender, which attacks male kids, and involves to motor disability in the property, leading to death around the second decade of life. It is caused by an alteration in the codifier gene of the protein dystropin, which maintains the integrity of the muscle fiber sarcolemma. The mdx mouse is used as an experimental model of DMD to investigate the skeletal and cardiac muscle fiber, because it presents a lot of similarities with the human carrier of the disease. In the DMD, the patients used to die due to respiratory failure. Since there was a treatment evolution with artificial mechanical ventilation techniques, the cardiac dysfunctions became important considering that from this moment on, most of the deaths started occurring because of a cardiac failure, resulting of a process followed by necrosis, inflammation, fibrosis involving to a serious cardiomyopathy. Several pharmacological strategies have been used to improve the cardiac function both in the DMD carrier and in the mdx one of the medications utilized is the deflazacort, a glucocorticóide of anti-inflammatory action, administrated during the whole life of the DMD carrier. Although the deflazacort effects upon the cardiac function in the human being and in the mouse mdx have been extensively studied just a little is know about the histopathological effects on the cardiac tissue. In this paper, the effect of the long term administration of deflazacort daily for 15 months, after they were sacrificed, had their hearts removed and frozen in liquid nitrogen for histological and morphological tissue further analysis. The heart of the mdx mice group treated with deflazacort was compared to the heart of the untreated mdx mice group. The myocardial fibrosis areas diminished significantly in comparison to the untreated group, in 40%. It was concluded that the long term treatment with deflazacort is effective to diminish the cardiac fibrosis progression the cardiomyopathy which cause the myocites cardiac necrosis, and therefore it is interesting to investigate the lipofuscin is a pigment related to the age, it is considered an aging biomarker. In this paper, the accumulation of lipofuscin in control animals C57BL10 and mdx with ages between 14 days and 21 months without any treatment was observed that they increase with the age, and from the 4 to the 6 months there was a raise in the lipofuscin accumulation. It was so, concluded, that the myocites cardiac functioning can be harmed even before the age of 8 months, and the accumulation of lipofuscin can mean a degeneration process which is more intensive in mdx / Mestrado / Biologia Celular / Mestre em Biologia Celular e Estrutural