Thesis (Ph. D.)--University of Wisconsin--Madison, 1955. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 70-75).

A rabbit lateral meniscectomy model was used to induce osteoarthritis. Separate studies were conducted to evaluate the progression of osteoarthritis and to identify possible biological markers. First, 21 male, New Zealand White rabbits were divided into 3 groups (n = 7 / group). A randomly selected left or right stifle underwent a lateral meniscectomy. The 3 groups were: corticosteroid administration, forced exercise and surgical control. An open field maze was used to assess mobility weekly. The rabbits were euthanitized 47 days after surgery. Histopathologic examination found that the lateral meniscectomy induced more severe lesions than in the non-surgical contralateral stifle. It also showed a significant sparing effect on erosion of cartilage in the corticosteroid group. The corticosteroid group, but not the exercise group, caused a significant increase in mobility (p = 0.008) compared to the surgical control. Secondly, synovial fluid was harvested from the 12 rabbits on days 0, 6, 26, 40, and 57 with surgery occurring on day 12. Trypan blue was used in the lavage fluid to estimate the volume of harvested synovial fluid. There was a significant increase in the volume harvested on day 26 (p < 0.001). Superoxide dismutase concentration in synovial fluid increased after surgery, although not significantly. These studies verify that the lateral meniscectomy model produce histopathologic lesions consistent with osteoarthritis. Furthermore, use of trypan blue appears to be a reliable concentration marker in a lavage sample to measure harvested synovial fluid. / Master of Science

A remoção cirúrgica da catarata é uma intervenção cada vez mais praticada na medicina veterinária e, com ela, diversos estudos têm sido realizados com o intuito de reduzir as possíveis complicações de seu pós-operatório, tal como a opacidade da cápsula posterior, alteração ocasionada principalmente pela diferenciação e migração das células do epitélio lenticular para a cápsula posterior da lente. O objetivo deste estudo foi analisar a morfologia da cápsula anterior e seu epitélio pela coloração de rotina com hematoxilina-eosina; a composição molecular da cápsula pelas técnicas histoquímicas de PAS (avaliação de proteoglicanos) e picrosirius red (avaliação de colágeno IV); além da expressão de morte celular nas células epiteliais pelas técnicas de imunofluorescência com Beclina 1 (autofagia) e Caspase 3 (apoptose), em cães idosos com catarata diabética e não diabética do tipo hipermadura, submetidos ao uso ou não de azul de tripano a 0,1 % durante a cirurgia de remoção da catarata por facoemulsificação. Para o estudo, foram estudadas as células epiteliais e cápsula anterior da lente de 27 cães, sendo 21 fêmeas e 6 machos, de 8 a 12 anos de idade (média = 9,6 anos), de diversas raças, que foram primeiramente divididos em 2 grupos: GC (catarata hipermadura) e GCD (catarata diabética) e, depois, subdivididos em outros 4 grupos: os que utilizaram o corante: GAND (catarata hipermadura com azul de tripano) e GAD (catarata diabética com azul de tripano); e os que não foram submetidos ao uso do corante: GND (catarata hipermadura sem azul de tripano) e GD (catarata diabética sem azul de tripano). Os resultados das análises realizadas observaram que ambas as amostras, tanto as provenientes das cataratas hipermaduras, quanto as diabéticas, sofrem alterações em igual intensidade apesar do diferente processo de formação, ou seja, observou-se que houve positividade na expressão de morte celular do epitélio por apoptose e autofagia em igual intensidade e independente da utilização de azul de tripano a 0,1 % em todos os grupos; que a composição molecular da cápsula quanto à quantidade de proteoglicanos e colágeno IV também é semelhante e que, apesar da cápsula apresentar-se mais espessada do que o normal, ainda preserva sua relação anatômica com o epitélio. Conclui-se, portanto, que as lesões ocasionadas pela formação da catarata diabética e hipermadura no epitélio e em sua membrana basal (cápsula) são semelhantes apesar da diferente fisiopatologia de formação, mas que o epitélio e a cápsula ainda preservam sua relação anatômica e morfologia. Com os resultados, também se pode sugerir que cães que apresentem cataratas destes tipos, devam apresentar menor risco de ocorrência de opacidade na cápsula posterior no pós cirúrgico da catarata / Nowadays, surgical removal of cataracts in dogs is being considered a common intervention. Along with that, many studies have been realized aiming to reduce its post operatory complications, such as the posterior lens capsule opacity, which occurs due to epithelium cells differentiation and migration to the posterior pole. The objective of this study was to evaluate, by morphometry (hematoxilin-eosin), histochemistry (PAS and picrosirius red) and immunofluorescence (Caspase 3 and Beclin 1) analysis: the morphological alterations of the lens anterior capsule and its epithelium; the molecular components of the extracellular matrix; and the expression of lens epithelium cellular death (by autophagy and apoptosis), in elderly diabetic and non diabetic dogs with cataracts, after phacoemulsification surgery utilizing or not 0,1 % trypan blue. Twenty seven dogs, including 21 female and 6 male dogs, with ages varying from 8 to 12 years old (mean = 9.6 yo), of different breeds were studied. Animals were first divided into 2 groups: GC (hypermature cataracts) and GCD (diabetic cataracts); then, subdivided in 4 different groups: GAND (non diabetic dogs with trypan blue), GAD (diabetic dogs with trypan blue), GND (non diabetic dogs without trypan blue) and finally, GD (diabetic dogs without trypan blue). Results showed that both types of studied samples (diabetic and hypermature ones), are equally altered besides its different pathophysiology, since no statistical significant differences were observed in both capsule and epithelium thickness, type IV collagen and proteoglycans concentration and also, in the epithelium cells autophagy and apoptosis, observed by Caspase 3 and Beclin 1 expression in every studied group. In addition, 0,1 % trypan blue was not capable to demonstrate its toxicity rate, as cells were already under the referred death processes. In conclusion, our findings suggests that, besides the differences in both, hypermature and diabetic cataracts, pathophysiologies of formation, serious and similar alterations are observed with the same intensity in the anterior lens capsule and its epithelium in dogs, what must probably reduce posterior lens capsule opacity post phacoemulsification surgery

Background and motivation: The event of neural Ca2+ overload is known to have several deleterious effects resulting in cell death caused by ischaemia, hypoglycaemia, hypoxia and several neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease and AIDS-related dementia. In vitro models for the investigation of the mechanisms involved in Ca2+ overload include brain slice preparations, neuronal cultures as well as acutely isolated neurons, mostly from the hippocampus and cortical brain areas. Additional models for investigating Ca2+ overload may bring about new knowledge to areas of the phenomenon that are still unresolved. Methodology: In this study, several theoretical Ca2+ overload-related interventions were combined aimed at inducing cell death in acutely isolated rat dorsal root ganglia. To elucidate the mechanism/s involved in the cell death observed following exposure to this intervention, the effects of several alterations to the intervention's composition were assessed. This examination was extended by the addition of several recognized and potential protective compounds to the intervention. Cell death was indicated by the trypan blue exclusion assay and recorded after 18 hours exposure to the interventions by counting live and dead neurons under a light microscope. Results and conclusions: The goal was to evaluate the possible application of dorsal root ganglia as a model for neural Ca2+ overload outside the brain. Since Ca2+w as required for cell death to be induced, it is concluded that the observed cell death was indeed primarily due to Ca2+ overload. Besides extracellular Ca2+, KC1-induced depolarization was also required for cell death to be induced, while the antagonists did not demonstrate significant protection against cell death. Based on the results, the mechanism of Ca2+ overload could not be defined beyond doubt, but the voltage activated Ca2+ channels are likely to be involved. / Thesis (M.Sc. (Physiology))--North-West University, Potchefstroom Campus, 2005.

Suspensions of pancreatic islet cells from ob/ob-mice were incubated with Trypan Blue. Microscope photometry showed that apparently viable cells excluded the dye completely, whereas the nuclei of non-viable cells accumulated Trypan Blue by a saturable process. Alloxan rapidly increased the permeability of the plasma membrane in mouse 3-cells; the exclusion of Trypan Blue is a valid and useful measure of islet cell viability following alloxan exposure. The diabetogenic action of alloxan may be mediated by hydroxyl radicals. In several biological systems hydroxyl radicals are formed by an iron-catalyzed reaction between superoxide anion radicals and hydrogen peroxide. To test whether this applies to alloxan diabetogenicity, the effects of superoxide dismutase, catalase, scavengers of hydroxyl radicals, and metal ion chelators were tested (a) in a cell-free radical-generating system and (b) on islets and islet-cells exposed to alloxan In vitro. The effect of longtime-circulating superoxide dismutase injected prior to alloxan was tested on mice in vivo. Luminol chemiluminescence was used to monitor alloxan-dependent radical production. Accumulation of 8^Rb+ and exclusion of Trypan Blue were used as cell viability criteria in isolated mouse islets and islet-cells. Blood glucose was determined to monitor the development of diabetes in living animals. Superoxide dismutase, catalase, scavengers of hydroxyl radicals, and metal ion chelators inhibited the alloxan-dependent chemiluminescence and decreased the toxic effects on Rb+ accumulation or Trypan Blue exclusion in islets and islet-cells. Superoxide dismutase, linked to polyethylene glycol and injected 12 hours before alloxan, largely prevented the development of alloxan diabetes. Alloxan toxicity _in vitro and in vivo seems to depend on the formation of superoxide radicals and hydrogen peroxide which in turn form the noxious hydroxyl radical via an iron-catalyzed Haber-Weiss reaction. As free radicals and hydrogen peroxide can be formed by other chemicals and during inflammation, and inflammation may accompany the outbreak of human diabetes, studies on the beneficiary effects of superoxide dismutase and other scavengers of free radicals in other forms of diabetes seem warranted. / <p>S. 1-38: sammanfattning, s. 39-74: Härtill 6 uppsatser</p> / digitalisering@umu

Background and motivation: The event of neural Ca2+ overload is known to have several deleterious effects resulting in cell death caused by ischaemia, hypoglycaemia, hypoxia and several neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease and AIDS-related dementia. In vitro models for the investigation of the mechanisms involved in Ca2+ overload include brain slice preparations, neuronal cultures as well as acutely isolated neurons, mostly from the hippocampus and cortical brain areas. Additional models for investigating Ca2+ overload may bring about new knowledge to areas of the phenomenon that are still unresolved. Methodology: In this study, several theoretical Ca2+ overload-related interventions were combined aimed at inducing cell death in acutely isolated rat dorsal root ganglia. To elucidate the mechanism/s involved in the cell death observed following exposure to this intervention, the effects of several alterations to the intervention's composition were assessed. This examination was extended by the addition of several recognized and potential protective compounds to the intervention. Cell death was indicated by the trypan blue exclusion assay and recorded after 18 hours exposure to the interventions by counting live and dead neurons under a light microscope. Results and conclusions: The goal was to evaluate the possible application of dorsal root ganglia as a model for neural Ca2+ overload outside the brain. Since Ca2+w as required for cell death to be induced, it is concluded that the observed cell death was indeed primarily due to Ca2+ overload. Besides extracellular Ca2+, KC1-induced depolarization was also required for cell death to be induced, while the antagonists did not demonstrate significant protection against cell death. Based on the results, the mechanism of Ca2+ overload could not be defined beyond doubt, but the voltage activated Ca2+ channels are likely to be involved. / Thesis (M.Sc. (Physiology))--North-West University, Potchefstroom Campus, 2005.

A remoção cirúrgica da catarata é uma intervenção cada vez mais praticada na medicina veterinária e, com ela, diversos estudos têm sido realizados com o intuito de reduzir as possíveis complicações de seu pós-operatório, tal como a opacidade da cápsula posterior, alteração ocasionada principalmente pela diferenciação e migração das células do epitélio lenticular para a cápsula posterior da lente. O objetivo deste estudo foi analisar a morfologia da cápsula anterior e seu epitélio pela coloração de rotina com hematoxilina-eosina; a composição molecular da cápsula pelas técnicas histoquímicas de PAS (avaliação de proteoglicanos) e picrosirius red (avaliação de colágeno IV); além da expressão de morte celular nas células epiteliais pelas técnicas de imunofluorescência com Beclina 1 (autofagia) e Caspase 3 (apoptose), em cães idosos com catarata diabética e não diabética do tipo hipermadura, submetidos ao uso ou não de azul de tripano a 0,1 % durante a cirurgia de remoção da catarata por facoemulsificação. Para o estudo, foram estudadas as células epiteliais e cápsula anterior da lente de 27 cães, sendo 21 fêmeas e 6 machos, de 8 a 12 anos de idade (média = 9,6 anos), de diversas raças, que foram primeiramente divididos em 2 grupos: GC (catarata hipermadura) e GCD (catarata diabética) e, depois, subdivididos em outros 4 grupos: os que utilizaram o corante: GAND (catarata hipermadura com azul de tripano) e GAD (catarata diabética com azul de tripano); e os que não foram submetidos ao uso do corante: GND (catarata hipermadura sem azul de tripano) e GD (catarata diabética sem azul de tripano). Os resultados das análises realizadas observaram que ambas as amostras, tanto as provenientes das cataratas hipermaduras, quanto as diabéticas, sofrem alterações em igual intensidade apesar do diferente processo de formação, ou seja, observou-se que houve positividade na expressão de morte celular do epitélio por apoptose e autofagia em igual intensidade e independente da utilização de azul de tripano a 0,1 % em todos os grupos; que a composição molecular da cápsula quanto à quantidade de proteoglicanos e colágeno IV também é semelhante e que, apesar da cápsula apresentar-se mais espessada do que o normal, ainda preserva sua relação anatômica com o epitélio. Conclui-se, portanto, que as lesões ocasionadas pela formação da catarata diabética e hipermadura no epitélio e em sua membrana basal (cápsula) são semelhantes apesar da diferente fisiopatologia de formação, mas que o epitélio e a cápsula ainda preservam sua relação anatômica e morfologia. Com os resultados, também se pode sugerir que cães que apresentem cataratas destes tipos, devam apresentar menor risco de ocorrência de opacidade na cápsula posterior no pós cirúrgico da catarata / Nowadays, surgical removal of cataracts in dogs is being considered a common intervention. Along with that, many studies have been realized aiming to reduce its post operatory complications, such as the posterior lens capsule opacity, which occurs due to epithelium cells differentiation and migration to the posterior pole. The objective of this study was to evaluate, by morphometry (hematoxilin-eosin), histochemistry (PAS and picrosirius red) and immunofluorescence (Caspase 3 and Beclin 1) analysis: the morphological alterations of the lens anterior capsule and its epithelium; the molecular components of the extracellular matrix; and the expression of lens epithelium cellular death (by autophagy and apoptosis), in elderly diabetic and non diabetic dogs with cataracts, after phacoemulsification surgery utilizing or not 0,1 % trypan blue. Twenty seven dogs, including 21 female and 6 male dogs, with ages varying from 8 to 12 years old (mean = 9.6 yo), of different breeds were studied. Animals were first divided into 2 groups: GC (hypermature cataracts) and GCD (diabetic cataracts); then, subdivided in 4 different groups: GAND (non diabetic dogs with trypan blue), GAD (diabetic dogs with trypan blue), GND (non diabetic dogs without trypan blue) and finally, GD (diabetic dogs without trypan blue). Results showed that both types of studied samples (diabetic and hypermature ones), are equally altered besides its different pathophysiology, since no statistical significant differences were observed in both capsule and epithelium thickness, type IV collagen and proteoglycans concentration and also, in the epithelium cells autophagy and apoptosis, observed by Caspase 3 and Beclin 1 expression in every studied group. In addition, 0,1 % trypan blue was not capable to demonstrate its toxicity rate, as cells were already under the referred death processes. In conclusion, our findings suggests that, besides the differences in both, hypermature and diabetic cataracts, pathophysiologies of formation, serious and similar alterations are observed with the same intensity in the anterior lens capsule and its epithelium in dogs, what must probably reduce posterior lens capsule opacity post phacoemulsification surgery

No description available.

O objetivo do estudo foi analisar a cápsula e o epitéilo subcapsular cristaliniano (ESC) de pacientes submetidos à capsulotomia curvilínea contínua (CCC) utilizando o corante azul de tripano (AT) a 0,1%, através de microscopia óptica (MO), da técnica TUNEL, de imunohistoquímica e de microscopia eletrônica transmissão (MET). Realizamos um estudo prospectivo, controlado e randomizado utilizando 30 amostras de cápsulas e ESC obtidos de pacientes após CCC durante cirurgia de facectomia. Essas amostras foram divididas em dois grupos (15 espécimes cada) um utilizando o AT (grupo experimental) no ato cirúrgico e o outro sem o uso do corante (grupo controle). As cápsulas e o ESC destes grupos foram fixados e processados para análises estruturais posteriores com técnicas de MO de rotina, técnica TUNEL para detecção de morte celular por apoptose, imunohistoquímica para analisar a expressão da beclina-1 (um marcador de morte celular por autofagia), além de análise ultraestrutural por meio da MET. Foram realizadas análises morfométricas das imagens de microscopia após captura e digitalização, utilizando o programa Image Pró Plus (Cybernetics®, USA). Foram encontrados resultados positivos para a expressão de morte celular por apoptose e por autofagia no grupo submetido ao uso do AT, enquanto que no grupo controle os resultados foram negativos. As análises através da MET do ESC mostraram alterações em células coradas com o AT, incluindo ruptura mitocondrial, dilatação das cisternas do retículo endoplasmático, aumento da elétrondensidade citoplasmática e nuclear, e alteração no perfil nuclear. Os resultados estatísticos obtidos pelo teste de Mann-Whitney a partir da morfometria obtida de micrografias, demonstraram diferenças morfológicas significativas entre os grupos estudados, tanto nas dimensões dos maiores eixos nucleares, quanto na relação perímetro/área do núcleo celular (p=0,03). Em relação à espessura da cápsula, do epitélio subcapsular e do conjunto dessas estruturas obtidas a partir da MO de rotina não houve diferença estatisticamente significativa entre os grupos (p= 0,1). O AT provoca no ESC toxicidade celular com sinais indicativos de morte celular. Observamos nos aspectos morfológicos e moleculares morte celular tanto pelo mecanismo de apoptose quanto de autofagia. A partir destes achados podemos sugerir que a ação do AT, talvez possa ajudar a prevenir ou reduzir a opacificação da cápsula posterior do cristalino no período pós-operatório das facectomias / The purpose of this study was to evaluate the effect of trypan blue (TB) 0.1% staining on lens epithelial cells (LECs) and capsules of patients undergoing capsulorhexis using routine optical microscopy (OM), TUNEL technique, immunohistochemistry and transmission electron microscopy (TEM). In a prospective controlled and randomized study we evaluated 30 samples of capsules with LECs obtained after capsulorhexis during cataract surgery. Samples were randomly assigned to one of two groups (15 specimens each), one submitted to TB (experimental group) during the surgery and the other without the dye (control group). The capsule and the LECs of both groups were fixed and processed for later structural analysis with routine optical microscopy, immunohistochemistry for beclin-1 expression (a marker of cell death by autophagy), and the TUNEL technique to detect apoptosis, in addition to ultra-structural analysis by TEM. Morphometrical analysis were performed by using the Image Pro Plus software (Cybernetics®, USA). In the TB-stained group we have found positive results for the expression of cell death by autophagy and apoptosis while in the control group the results were negative. Analysis of LEC by TEM showed abnormalities in TB-stained cells including mitochondrial disruption, dilation of the endoplasmic reticulum cisterns, increased cytoplasmic and nuclear electron density and abnormalities in the nuclear profile. Statistical analysis using the Mann-Whitney test on morphometric data from micrographies showed significant morphologic differences between the two groups, both regarding longest nuclear axis difference and the ratio between the total nuclear perimeter and the cell area (p=0.03). No statistically significant difference was observed in capsule thickness, the LEC and the grouping of these two structures obtained from routine OM (p=0.1). Trypan blue is toxic to LECs, and cause abnormalities indicative of cell death. We observed molecular and morphologic aspects of cell death both by the mechanism of apoptosis and autophagy. Our findings lend support to the hypothesis that staining with 0.1% TB can help prevent or reduce the incidence of posterior capsule opacification following cataract surgery

O objetivo do estudo foi analisar a cápsula e o epitéilo subcapsular cristaliniano (ESC) de pacientes submetidos à capsulotomia curvilínea contínua (CCC) utilizando o corante azul de tripano (AT) a 0,1%, através de microscopia óptica (MO), da técnica TUNEL, de imunohistoquímica e de microscopia eletrônica transmissão (MET). Realizamos um estudo prospectivo, controlado e randomizado utilizando 30 amostras de cápsulas e ESC obtidos de pacientes após CCC durante cirurgia de facectomia. Essas amostras foram divididas em dois grupos (15 espécimes cada) um utilizando o AT (grupo experimental) no ato cirúrgico e o outro sem o uso do corante (grupo controle). As cápsulas e o ESC destes grupos foram fixados e processados para análises estruturais posteriores com técnicas de MO de rotina, técnica TUNEL para detecção de morte celular por apoptose, imunohistoquímica para analisar a expressão da beclina-1 (um marcador de morte celular por autofagia), além de análise ultraestrutural por meio da MET. Foram realizadas análises morfométricas das imagens de microscopia após captura e digitalização, utilizando o programa Image Pró Plus (Cybernetics®, USA). Foram encontrados resultados positivos para a expressão de morte celular por apoptose e por autofagia no grupo submetido ao uso do AT, enquanto que no grupo controle os resultados foram negativos. As análises através da MET do ESC mostraram alterações em células coradas com o AT, incluindo ruptura mitocondrial, dilatação das cisternas do retículo endoplasmático, aumento da elétrondensidade citoplasmática e nuclear, e alteração no perfil nuclear. Os resultados estatísticos obtidos pelo teste de Mann-Whitney a partir da morfometria obtida de micrografias, demonstraram diferenças morfológicas significativas entre os grupos estudados, tanto nas dimensões dos maiores eixos nucleares, quanto na relação perímetro/área do núcleo celular (p=0,03). Em relação à espessura da cápsula, do epitélio subcapsular e do conjunto dessas estruturas obtidas a partir da MO de rotina não houve diferença estatisticamente significativa entre os grupos (p= 0,1). O AT provoca no ESC toxicidade celular com sinais indicativos de morte celular. Observamos nos aspectos morfológicos e moleculares morte celular tanto pelo mecanismo de apoptose quanto de autofagia. A partir destes achados podemos sugerir que a ação do AT, talvez possa ajudar a prevenir ou reduzir a opacificação da cápsula posterior do cristalino no período pós-operatório das facectomias / The purpose of this study was to evaluate the effect of trypan blue (TB) 0.1% staining on lens epithelial cells (LECs) and capsules of patients undergoing capsulorhexis using routine optical microscopy (OM), TUNEL technique, immunohistochemistry and transmission electron microscopy (TEM). In a prospective controlled and randomized study we evaluated 30 samples of capsules with LECs obtained after capsulorhexis during cataract surgery. Samples were randomly assigned to one of two groups (15 specimens each), one submitted to TB (experimental group) during the surgery and the other without the dye (control group). The capsule and the LECs of both groups were fixed and processed for later structural analysis with routine optical microscopy, immunohistochemistry for beclin-1 expression (a marker of cell death by autophagy), and the TUNEL technique to detect apoptosis, in addition to ultra-structural analysis by TEM. Morphometrical analysis were performed by using the Image Pro Plus software (Cybernetics®, USA). In the TB-stained group we have found positive results for the expression of cell death by autophagy and apoptosis while in the control group the results were negative. Analysis of LEC by TEM showed abnormalities in TB-stained cells including mitochondrial disruption, dilation of the endoplasmic reticulum cisterns, increased cytoplasmic and nuclear electron density and abnormalities in the nuclear profile. Statistical analysis using the Mann-Whitney test on morphometric data from micrographies showed significant morphologic differences between the two groups, both regarding longest nuclear axis difference and the ratio between the total nuclear perimeter and the cell area (p=0.03). No statistically significant difference was observed in capsule thickness, the LEC and the grouping of these two structures obtained from routine OM (p=0.1). Trypan blue is toxic to LECs, and cause abnormalities indicative of cell death. We observed molecular and morphologic aspects of cell death both by the mechanism of apoptosis and autophagy. Our findings lend support to the hypothesis that staining with 0.1% TB can help prevent or reduce the incidence of posterior capsule opacification following cataract surgery