Ho Yi-Fong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 120-131). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgements --- p.iv / Table of Contents --- p.v / List of Figures --- p.ix / List of Tables --- p.xi / Abbreviations --- p.xii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Corticosteroids in ophthalmology --- p.1 / Chapter 1.1.1 --- History of the clinical use of corticosteroids --- p.1 / Chapter 1.1.2 --- Administration --- p.1 / Chapter 1.1.3 --- General biological effects of corticosteroids --- p.4 / Chapter 1.1.4 --- Application of corticosteroids in ocular diseases --- p.5 / Chapter 1.1.5 --- Side effects of ocular corticosteroid treatment --- p.6 / Chapter 1.1.6 --- General introduction to commonly used corticosteroids in ophthalmology --- p.6 / Chapter 1.1.6.1 --- Hydrocortisone --- p.6 / Chapter 1.1.6.2 --- Dexamethasone --- p.7 / Chapter 1.1.6.3 --- Triamcinolone --- p.7 / Chapter 1.1.6.4. --- Chemical structures and relative anti-inflammatory potencies --- p.8 / Chapter 1.1.7 --- Cytotoxicity of triamcinolone --- p.12 / Chapter 1.2 --- Retinal ganglion cells --- p.13 / Chapter 1.2.1 --- Basic structures of the eye --- p.13 / Chapter 1.2.2 --- Anatomical structure of retina --- p.13 / Chapter 1.2.3 --- Functions of retinal ganglion cells --- p.18 / Chapter 1.2.4 --- Culture models to study RGCs --- p.20 / Chapter 1.3 --- Aim of study --- p.25 / Chapter Chapter 2 --- Methodology --- p.26 / Chapter 2.1 --- Intravitreal injection of TA (IVTA) --- p.26 / Chapter 2.1.1 --- Materials --- p.26 / Chapter 2.1.1.1 --- Animals --- p.26 / Chapter 2.1.1.2 --- Chemicals --- p.26 / Chapter 2.1.1.3 --- Instruments --- p.26 / Chapter 2.1.2 --- Procedures --- p.26 / Chapter 2.2 --- Peripheral Nerve - Optic Nerve Grafting (PN-ON) Procedure --- p.27 / Chapter 2.3 --- Retrograde Labeling of regenerating RGCs --- p.27 / Chapter 2.3.1 --- Materials --- p.27 / Chapter 2.3.2 --- Procedures --- p.27 / Chapter 2.3.3 --- Statistical analysis --- p.28 / Chapter 2.4 --- Immunohistochemistry --- p.28 / Chapter 2.4.1 --- Materials --- p.28 / Chapter 2.4.2 --- Procedures --- p.29 / Chapter 2.4.3 --- Statistical analysis --- p.29 / Chapter 2.5 --- Histology --- p.29 / Chapter 2.5.1 --- Materials --- p.29 / Chapter 2.5.2 --- Procedures --- p.29 / Chapter 2.6 --- Primary rat retinal ganglion cell culture --- p.30 / Chapter 2.6.1 --- Materials --- p.30 / Chapter 2.6.1.1 --- Animals --- p.30 / Chapter 2.6.1.2 --- Chemicals --- p.30 / Chapter 2.6.1.3 --- Solutions and buffers --- p.30 / Chapter 2.6.1.4 --- Instruments --- p.31 / Chapter 2.6.2 --- Preparations --- p.31 / Chapter 2.6.2.1 --- Working media --- p.31 / Chapter 2.6.2.2 --- Plate coating --- p.32 / Chapter 2.6.3 --- Cell culture process --- p.32 / Chapter 2.6.3.1 --- Dissection of retinal tissues --- p.32 / Chapter 2.6.3.2 --- Purification of RGCs --- p.33 / Chapter 2.6.3.3 --- Culture condition and cell seeding --- p.34 / Chapter 2.7 --- Corticosteroid treatment --- p.34 / Chapter 2.7.1 --- Materials --- p.34 / Chapter 2.7.2 --- Preparations --- p.34 / Chapter 2.7.3 --- Treatment --- p.35 / Chapter 2.8 --- Cell viability assay and morphometric study --- p.36 / Chapter 2.8.1 --- Materials --- p.36 / Chapter 2.8.2 --- Calcein-AM staining --- p.36 / Chapter 2.8.3 --- Cell viability --- p.37 / Chapter 2.8.4 --- Morphometry study --- p.37 / Chapter 2.9 --- TUNEL Assay --- p.38 / Chapter 2.9.1 --- Materials --- p.38 / Chapter 2.9.2 --- Procedure --- p.38 / Chapter 2.9.3 --- Statistical analysis --- p.39 / Chapter 2.10 --- Quantitative Reverse transcription - Polymerase Chain Reaction (qRT-PCR) --- p.39 / Chapter 2.10.1 --- Materials --- p.39 / Chapter 2.10.1.1 --- "Chemicals, reagents, and kits" --- p.39 / Chapter 2.10.1.2 --- Primers --- p.40 / Chapter 2.10.1.3 --- Equipment --- p.41 / Chapter 2.10.1.4 --- Software --- p.41 / Chapter 2.10.2 --- Procedures --- p.41 / Chapter 2.10.2.1 --- Cell collection and RNA isolation --- p.41 / Chapter 2.10.2.2 --- Reverse Transcription --- p.42 / Chapter 2.10.2.3 --- Real-time PCR --- p.43 / Chapter 2.10.3 --- Statistical analysis --- p.43 / Chapter 2.11 --- Western blotting --- p.44 / Chapter 2.11.1 --- Sample preparation --- p.44 / Chapter 2.11.1.1 --- Materials --- p.44 / Chapter 2.11.1.1.1 --- Chemicals and materials --- p.44 / Chapter 2.11.1.1.2 --- Equipment --- p.44 / Chapter 2.11.1.2 --- Procedures --- p.44 / Chapter 2.11.2 --- Protein assay --- p.45 / Chapter 2.11.2.1 --- Materials --- p.45 / Chapter 2.11.2.1.1 --- Chemicals and materials --- p.45 / Chapter 2.11.2.1.2 --- Equipment and software --- p.46 / Chapter 2.11.2.2 --- Procedures --- p.46 / Chapter 2.11.3 --- SDS-polyacrylamide gel electrophoresis (SDS-PAGE) --- p.46 / Chapter 2.11.3.1 --- Materials --- p.46 / Chapter 2.11.3.1.1 --- Chemicals and reagents --- p.46 / Chapter 2.11.3.1.2 --- Equipment --- p.46 / Chapter 2.11.3.1.3 --- Solutions and buffers --- p.47 / Chapter 2.11.3.2 --- Gel preparation --- p.48 / Chapter 2.11.3.3 --- Electrophoresis --- p.49 / Chapter 2.11.3.4 --- Transblotting (semi-dry transfer) --- p.49 / Chapter 2.11.3.5 --- Band visualization --- p.49 / Chapter 2.11.4 --- Immunostaining --- p.50 / Chapter 2.11.4.1 --- Materials --- p.50 / Chapter 2.11.4.1.1 --- Antibodies --- p.50 / Chapter 2.11.4.1.2 --- Chemicals and reagents --- p.50 / Chapter 2.11.4.1.3 --- Equipment --- p.50 / Chapter 2.11.4.2 --- Procedures --- p.50 / Chapter 2.12 --- Gas-chromatography-electron-capture negative-ion mass spectrometry (GC-NCI-MS) --- p.51 / Chapter 2.12.1 --- Standard and reagents --- p.51 / Chapter 2.12.2 --- Chromatography and mass spectrometry --- p.52 / Chapter 2.12.3 --- Sample collection --- p.52 / Chapter 2.12.3 --- Standard and sample preparation --- p.53 / Chapter 2.12.4 --- Validation --- p.54 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Effect of triamcinolone on RGCs in vivo --- p.55 / Chapter 3.2 --- Cell viability of RGCs after IVTA plus PN-ON grafting --- p.55 / Chapter 3.3 --- Abnormal retinal morphology under different IVTA conditions --- p.59 / Chapter 3.4 --- Cell viability assay --- p.66 / Chapter 3.4.1 --- Effects of triamcinolone on RGC viability --- p.66 / Chapter 3.4.2 --- Effects of dexamethasone on RGC viability --- p.68 / Chapter 3.4.3 --- Effects of hydrocortisone on RGC viability --- p.70 / Chapter 3.4.4 --- Effects of filtered fraction of triamcinolone on RGC viability --- p.72 / Chapter 3.5 --- Morphometric study analysis of RGCs --- p.74 / Chapter 3.5.1 --- Percentage of RGCs showing neurite outgrowth --- p.74 / Chapter 3.5.2 --- Average neurite length --- p.77 / Chapter 3.5.3 --- Neurite spanning area --- p.77 / Chapter 3.5.4 --- Neurite count --- p.80 / Chapter 3.5.5 --- Neurite branching --- p.83 / Chapter 3.6 --- Determination of concentration of TA in culture media by GC-NCI-MS --- p.85 / Chapter 3.7 --- TUNEL assay --- p.90 / Chapter 3.8 --- Real-time quantitative Reverse transcription - Polymerase Chain Reaction --- p.93 / Chapter 3.9 --- Western blot --- p.99 / Chapter Chapter 4 --- Discussion --- p.102 / Chapter Chapter 5 --- References --- p.120

Glucocorticoids are known to affect metabolic activities of cells. The mechanism of glucocorticoid actions in adult human dermal and mouse L-929 fibroblasts have yet to be fully ascertained. This study endeavors to examine the effects of one glucocorticoid, triamcinolone acetonide, on cellular proliferation and collagen synthesis and to compare such effects in the human and mouse cell lines. Cellular proliferation and collagen synthesis are analyzed and quantitated by cell counts and selective digestion of the protein by bacterial collagenase, respectively. Further analysis of collagen synthesis is provided by polyacrylamide gel electrophoresis. One-tenth triamcinolone acetonide per ml suppresses cellular proliferation of mouse L-929 fibroblasts. Proline incorporation into total and collagenase-sensitive protein is enhanced in the cell layer; that of medium is altered inconsistently. Polyacrylamide gel electrophoresis of proteins treated with pepsin show the abolition of total and collagenase-sensitive protein in the cell layer. Aberrations in hydroxylation and/or deformation in physical structure of protein may confer greater susceptibility to pepsin digestion. Cellular proliferation and proline incorporation into total and collagenase-sensitive protein of adult human dermal fibroblasts are affected inconsistently by the same dose of triamcinolone acetonide. Except for the consistent suppression of cellular proliferation in the murine L-929 fibroblasts by triamcinolone acetonide, all observations pertaining to human dermal fibroblasts are incompatible with those obtained by other workers. Manipulation of culture conditions and glucocorticoid treatment dictate, to a large extent, the kind of responses observed. This could account for the wide variability and frequent contradictory findings reported in the literature. / Pharmaceutical Sciences, Faculty of / Graduate

Zheng Tingting. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 98-124). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgements --- p.iv / Table of Contents --- p.vi / List of Tables --- p.viii / List of Figures --- p.ix / Abbreviations --- p.xi / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Triamcinolone acetonide(TA) --- p.1 / Chapter 1.1.1 --- Application in ophthalmology --- p.1 / Chapter 1.1.2 --- Mechanism of anti-inflammatory effect --- p.2 / Chapter 1.1.3 --- Side effects of TA --- p.4 / Chapter 1.1.4 --- Toxicity of TA --- p.5 / Chapter 1.2 --- Retinal pigment epithelial (RPE) cell --- p.6 / Chapter 1.3 --- Mechanism of cell death --- p.8 / Chapter 1.3.1 --- Apoptosis --- p.9 / Chapter 1.3.2 --- caspase --- p.11 / Chapter 1.3.3 --- Mitogen-activated protein kinase (MAPK) --- p.13 / Chapter 1.3.4 --- Activator Protein-1 (AP-1) --- p.15 / Chapter 1.4 --- Potassium channel (K+）） --- p.15 / Chapter 1.4.1 --- Molecular structure of KAtp channel --- p.16 / Chapter 1.4.2 --- Regulation of Katp channel --- p.17 / Chapter 1.4.3 --- Pinacidil --- p.18 / Chapter 1.5 --- Calcium channel --- p.20 / Chapter 1.5.1 --- VDCCs and subtypes --- p.21 / Chapter 1.5.2 --- Calcium channel blocker --- p.22 / Chapter 1.5.3 --- Verapamil --- p.23 / Chapter 1.6 --- Study objectives --- p.25 / Chapter Chapter 2 --- Methodology --- p.37 / Chapter 2.1 --- Cell biology --- p.37 / Chapter 2.1.1 --- Materials --- p.37 / Chapter 2.1.1.1 --- Culture related material --- p.37 / Chapter 2.1.1.2 --- Drugs --- p.37 / Chapter 2.1.1.3 --- Cell line and instrument --- p.37 / Chapter 2.1.2 --- Preparations --- p.38 / Chapter 2.1.2.1 --- Working medium --- p.38 / Chapter 2.1.2.2 --- Drugs --- p.38 / Chapter 2.1.2.3 --- MTT solution --- p.39 / Chapter 2.1.3 --- Cell culture and treatment process --- p.40 / Chapter 2.1.3.1 --- Seed cell --- p.40 / Chapter 2.1.3.2 --- Treatment --- p.40 / Chapter 2.1.4 --- MTT-Cell Proliferation Assay --- p.41 / Chapter 2.2 --- Molecular biology --- p.42 / Chapter 2.2.1 --- Materials --- p.42 / Chapter 2.2.1.1 --- "Chemicals, reagents, and kits" --- p.42 / Chapter 2.2.1.2 --- Solutions and Buffers --- p.42 / Chapter 2.2.1.3 --- Primers and Enzymes --- p.43 / Chapter 2.2.1.4 --- Equipment --- p.43 / Chapter 2.2.1.5 --- Software --- p.43 / Chapter 2.2.2 --- Reverse transcription 226}0ؤ Polymerase Chain Reaction (RT-PCR) --- p.44 / Chapter 2.2.2.1 --- Cell collection and RNA Isolation --- p.44 / Chapter 2.2.2.2 --- Reverse Transcription (RT) --- p.45 / Chapter 2.2.2.3 --- PCR Reaction --- p.46 / Chapter 2.3 --- Immunocytochemistry --- p.48 / Chapter 2.3.1 --- Materials and instrumentation --- p.49 / Chapter 2.3.1.1 --- Antibodies and Equipment --- p.49 / Chapter 2.3.1.2 --- Chemicals and other useful items --- p.49 / Chapter 2.3.2 --- Preparations --- p.50 / Chapter 2.3.2.1 --- Preparation of coverslips --- p.50 / Chapter 2.3.2.2 --- Prepations of solutions --- p.50 / Chapter 2.3.3 --- Procedures --- p.51 / Chapter 2.4 --- Expression of results and statistics --- p.52 / Chapter Chapter 3 --- Results --- p.57 / Chapter 3.1 --- Effects of TA on RPE cell culture --- p.57 / Chapter 3.1.1 --- Cell morphology --- p.57 / Chapter 3.1.2 --- MTT assay --- p.57 / Chapter 3.1.3 --- Gene expressions --- p.57 / Chapter 3.2 --- Effects of PIN/VP on TA treated RPE cells --- p.58 / Chapter 3.2.1 --- MTT assay --- p.58 / Chapter 3.2.2 --- Gene expression --- p.59 / Chapter 3.2.2.1 --- Expression of housekeeping gene --- p.59 / Chapter 3.2.2.2 --- Expression of apoptosis-related gene --- p.59 / Chapter 3.2.2.3 --- Expression of early-response genes --- p.60 / Chapter 3.2.3 --- Immunofluorescence --- p.61 / Chapter Chapter 4 --- Discussion --- p.86 / Chapter Chapter 5 --- References --- p.98

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Os glicocorticóides são muito utilizados na clínica veterinária por serem potentes inibidores da resposta inflamatória. São encontrados em muitas preparações tópicas, sozinhos ou combinados com outros medicamentos e estão presentes, principalmente, em produtos destinados ao uso otológico. Mesmo quando aplicados topicamente, sabe-se que existe certo grau de absorção. Com a disponibilidade de novos glicocorticóides sintéticos mais potentes, vem sendo sugerido recentemente, a ocorrência de hiperadrenocorticismo iatrogênico em pequenos animais. Com a finalidade de se testar a hipótese que a utilização de preparações otológicas contendo glicocorticóides podem ocorrer em efeitos sistêmicos, o presente estudo avaliou três antiinflamatórios esteroidais comumente utilizados no tratamento de otite em cães e gatos, quais sejam a hidrocortisona, a betametasona e a triancinolona. Para tanto, ratos foram tratados tópicamente, via auricular, a cada 12 horas, durante 7, 14 e 21 dias, com os fármacos descritos acima. Utilizamos o edema de pata induzido pela injeção de carragenina como forma de avaliar a existência de níveis efetivos de antiinflamatórios circulantes. Ainda, foram avaliados vários parâmetros tais como variação de peso corpóreo, peso relativo de baço e adrenais, hemograma completo, índice de proliferação de linfócitos e fagocitose e burst oxidativo realizados por neutrófilos circulantes. Além disso, foi avaliado o nível de corticosterona plasmática nos animais tratados com glicocorticóides tópicos. O tratamento tópico com antiinflamatórios esteroidais levou a redução no volume de edema inflamatório, mais evidente com a triancinolona. De forma geral, houve queda no ganho, ou mesmo perda de peso corpóreo durante o tratamento tópico com os antiinflamatórios esteroidais empregados. Em alguns casos, essa redução de peso corpóreo foi acompanhada de diminuição do peso relativo do baço e adrenais, particularmente com a betametasona. A principal alteração observada no hemograma foi leucopenia, mais evidente com a triancinolona. Houve redução dos níveis plasmáticos de corticosterona, mais duradoura com a betametasona. Os tratamentos tópicos causaram discreta redução na intensidade e percentual de fagocitose realizada por neutrófilos circulantes, bem como na produção de espécies reativas de oxigênio. O estudo aponta que glicocorticóides tópicos comumente empregados na terapêutica veterinária podem ser absorvidos, na dependência da molécula empregada e de seu tempo de utilização, causando alterações sistêmicas relevantes já após 7 dias de tratamento. Sugerimos uma maior cautela e acompanhamento veterinário dos pacientes nos casos que haja necessidade de emprego de tais medicamentos por períodos prolongados. / Glucocorticoids are commonly employed in veterinary practice because they are potent inhibitors of the inflammatory response. They are present in many topical formulations, alone or combined with other active compounds, particularly in otic ointments. Even when applied topically, certain degree of absortion is known to occur. Following the availability of novel, more potent synthetic steroidal anti-inflammatory drugs, cases of iatrogenic hyperadrenocorticism have been suggested. Aiming on testing the hypothesis that glucocorticoid-containing otic ointments may lead to systemic effects, we employed three different synthetic glucocorticoids widely used for the treatment of otitis in small animals (hydrocortisone, betamethasone, and triamcinolone) our study Rats were treated topically on their ears, every 12 hours, for 7, 14, or 21 days with the drugs described above. We first employed carrageenan-induced paw edema in order to evaluate the presence of effective levels of circulating anti-inflammatory drugs. We also evaluated several parameters such as change in body weight, relative weight of spleen and adrenal glands, complete hemogram, proliferation index of lymphocytes, and phagocytosis and oxidative burst performed by circulating neutrophils. Moreover, we assessed the plasmatic levels of corticosterone in rats treated topically with anti-inflammatory steroids. Topical treatment led to a reduction on the volume of the inflammatory edema induced by carrageenan, more evident following triamcinolone. Altogether, rats treated with these molecules ceased to gain, or lost weight on the course of the experiment. In some cases, such reduction was accompanied by a decrease on the relative weight of the spleen and adrenal glands, particularly after betamethasone. The most commonly observed change in the hemogram was leucopenia, evident following triamcinolone. We found reduced levels of circulating corticosterone, more persistent after betamethasone. Topically applied glucocorticoids caused a discrete reduction in the intensity and percentage of phagocytosis by circulating neutrophils, and a reduction on the oxidative burst performed by these cells. This study shows that topical steroidal anti-inflammatory drugs, so commonly employed in veterinary practice, can be absorbed, depending on the molecule and duration of treatment, leading to systemic effects, which are already relevant after 7 days. Based on our findings, we suggest caution and a close follow-up by a qualified veterinarian whenever a long-term therapy based on these molecules and route of administration is required.

Os glicocorticóides são muito utilizados na clínica veterinária por serem potentes inibidores da resposta inflamatória. São encontrados em muitas preparações tópicas, sozinhos ou combinados com outros medicamentos e estão presentes, principalmente, em produtos destinados ao uso otológico. Mesmo quando aplicados topicamente, sabe-se que existe certo grau de absorção. Com a disponibilidade de novos glicocorticóides sintéticos mais potentes, vem sendo sugerido recentemente, a ocorrência de hiperadrenocorticismo iatrogênico em pequenos animais. Com a finalidade de se testar a hipótese que a utilização de preparações otológicas contendo glicocorticóides podem ocorrer em efeitos sistêmicos, o presente estudo avaliou três antiinflamatórios esteroidais comumente utilizados no tratamento de otite em cães e gatos, quais sejam a hidrocortisona, a betametasona e a triancinolona. Para tanto, ratos foram tratados tópicamente, via auricular, a cada 12 horas, durante 7, 14 e 21 dias, com os fármacos descritos acima. Utilizamos o edema de pata induzido pela injeção de carragenina como forma de avaliar a existência de níveis efetivos de antiinflamatórios circulantes. Ainda, foram avaliados vários parâmetros tais como variação de peso corpóreo, peso relativo de baço e adrenais, hemograma completo, índice de proliferação de linfócitos e fagocitose e burst oxidativo realizados por neutrófilos circulantes. Além disso, foi avaliado o nível de corticosterona plasmática nos animais tratados com glicocorticóides tópicos. O tratamento tópico com antiinflamatórios esteroidais levou a redução no volume de edema inflamatório, mais evidente com a triancinolona. De forma geral, houve queda no ganho, ou mesmo perda de peso corpóreo durante o tratamento tópico com os antiinflamatórios esteroidais empregados. Em alguns casos, essa redução de peso corpóreo foi acompanhada de diminuição do peso relativo do baço e adrenais, particularmente com a betametasona. A principal alteração observada no hemograma foi leucopenia, mais evidente com a triancinolona. Houve redução dos níveis plasmáticos de corticosterona, mais duradoura com a betametasona. Os tratamentos tópicos causaram discreta redução na intensidade e percentual de fagocitose realizada por neutrófilos circulantes, bem como na produção de espécies reativas de oxigênio. O estudo aponta que glicocorticóides tópicos comumente empregados na terapêutica veterinária podem ser absorvidos, na dependência da molécula empregada e de seu tempo de utilização, causando alterações sistêmicas relevantes já após 7 dias de tratamento. Sugerimos uma maior cautela e acompanhamento veterinário dos pacientes nos casos que haja necessidade de emprego de tais medicamentos por períodos prolongados. / Glucocorticoids are commonly employed in veterinary practice because they are potent inhibitors of the inflammatory response. They are present in many topical formulations, alone or combined with other active compounds, particularly in otic ointments. Even when applied topically, certain degree of absortion is known to occur. Following the availability of novel, more potent synthetic steroidal anti-inflammatory drugs, cases of iatrogenic hyperadrenocorticism have been suggested. Aiming on testing the hypothesis that glucocorticoid-containing otic ointments may lead to systemic effects, we employed three different synthetic glucocorticoids widely used for the treatment of otitis in small animals (hydrocortisone, betamethasone, and triamcinolone) our study Rats were treated topically on their ears, every 12 hours, for 7, 14, or 21 days with the drugs described above. We first employed carrageenan-induced paw edema in order to evaluate the presence of effective levels of circulating anti-inflammatory drugs. We also evaluated several parameters such as change in body weight, relative weight of spleen and adrenal glands, complete hemogram, proliferation index of lymphocytes, and phagocytosis and oxidative burst performed by circulating neutrophils. Moreover, we assessed the plasmatic levels of corticosterone in rats treated topically with anti-inflammatory steroids. Topical treatment led to a reduction on the volume of the inflammatory edema induced by carrageenan, more evident following triamcinolone. Altogether, rats treated with these molecules ceased to gain, or lost weight on the course of the experiment. In some cases, such reduction was accompanied by a decrease on the relative weight of the spleen and adrenal glands, particularly after betamethasone. The most commonly observed change in the hemogram was leucopenia, evident following triamcinolone. We found reduced levels of circulating corticosterone, more persistent after betamethasone. Topically applied glucocorticoids caused a discrete reduction in the intensity and percentage of phagocytosis by circulating neutrophils, and a reduction on the oxidative burst performed by these cells. This study shows that topical steroidal anti-inflammatory drugs, so commonly employed in veterinary practice, can be absorbed, depending on the molecule and duration of treatment, leading to systemic effects, which are already relevant after 7 days. Based on our findings, we suggest caution and a close follow-up by a qualified veterinarian whenever a long-term therapy based on these molecules and route of administration is required.

Objetivo: Avaliar a eficiência da Triancinolona Acetonida como agente modulador da resposta inflamatória e cicatricial em coelhos submetidos a cirurgia de músculo extra-ocular. Método: Foi realizado estudo prospectivo, mascarado, em dois estádios. No primeiro estádio, dez coelhos foram submetidos a retrocesso do músculo reto superior em ambos os olhos. Aplicouse, em um deles, 0,15 cc de Triancinolona Acetonida (40mg/cc) nos tecidos circunjacentes ao local de reinserção muscular e, como controle, 0,15cc de solução de cloreto de sódio a 0,9% no local equivalente no olho contra-lateral. Quinze dias após, cinco coelhos foram submetidos a exenteração das órbitas e os restantes dos animais tiveram o mesmo procedimento realizado após trinta dias. O material do sítio de reinserção muscular foi avaliado por meio de análise histopatológica qualitativa e quantitativa (morfometria). No segundo estádio, com incrementação da agressão cirúrgica, dezesseis coelhos foram submetidos aos mesmos procedimentos com exenteração das órbitas após quinze dias, e posterior análise histopatológica dos tecidos. Resultados: Houve efeito inibitório sobre a intensidade da resposta inflamatória nos olhos tratados em comparação com os olhos controle. Conclusão: Nas condições de realização do presente estudo o uso per-operatório da triancinolona acetonida foi efetivo no controle da resposta inflamatória em olhos de coelhos submetidos a cirurgia de músculo extra-ocular. / Purpose: To evaluate the efficiency of triamcinolone acetonide (TRI) in limiting the postoperative inflammatory response and scarring following strabismus surgery. Methods: A prospective, two-stage, masked, controlled trial was conducted. In the first stage, the inflammatory response at the extraocular reattachment site was analyzed following superior rectus recession in ten rabbits. One eye had 0,15 cc of triamcinolone acetonide (40mg/cc) applied around the new insertion site and, similarly, 0,15 cc of isotonic saline solution (0,9%) was applied to the fellow eye following the same procedure, thus serving as a control. Fifteen days later, orbital exenteration was performed in five rabbits and the remaining five were exenterated thirty days later. The reattachment site tissues were submitted to qualitative and quantitative histological examinations. In the second stage 16 rabbits were submitted to amplified surgical trauma, after which the aforementioned steps were also carried out. Granuloma total area at the extraocular muscle reattachment sites of control and treated eyes were compared. Results: There was an inhibitory effect of TRI on the inflammatory response of treated eyes as compared to control eyes. Conclusions: TRI was effective in controlling the postoperative inflammatory response in rabbit eyes submitted to traumatic recession of the superior rectus muscle.

O tratamento farmacológico de patologias bucais é conduzido, geralmente, por via de adminis-tração local. No entanto, devido ao pouco tempo de permanência do fármaco no local de ação, esse tratamento pode ser bastante comprometido. Assim, este trabalho teve por objetivo o de-senvolvimento de formas farmacêuticas que proporcionem a liberação local de triancinolona na cavidade oral. Foram produzidos filmes e comprimidos mucoadesivos a partir de polímeros naturais como gelana e pectina. Os filmes bucais foram preparados por meio de evaporação do solvente (solvent casting) utilizando diferentes quantidades de polímeros. As matérias-primas e os filmes foram caracterizados fisico quimicamente utilizando espectroscopia vibracional (in-fravermelho com transformada de Fourier e Raman) e difração de raios X. As propiedades físicas e mecânicas dos filmes também foram avaliadas. Além disso, realizou-se os ensaios de mucoadesividade e de dissolução do fármaco. Os comprimidos foram preparados por com-pressão direta usando como base os polímeros naturais. Diferentes parâmetros em relação as misturas e as formulações foram avaliados tais como as propriedades de fluxo dos pós consti-tuintes, peso médio, dureza, friabilidade e desintegração. Em relação aos filmes bucais, estes foram obtidos com sucesso através de um método simples, sem a utilização de agentes reticu-lantes, ácidos ou solventes orgânicos. Todos apresentaram bons resultados nas propriedades avaliadas, no entanto as formulações com quantidades intermediarias de polímeros foram as melhores. Dentre as formulações de comprimidos preparadas, apenas 4 apresentaram boas ca-racterísticas, no entanto, os resultados dos ensaios de dissolução mostraram que estas formula-ções têm capacidade de agir como sistema de liberação controlada de fármacos. / Pharmacological treatment of oral pathologies is usually conducted by local administration. However, due to the short time the drug stays in the site of action, this treatment can be quite compromised. Thus, the objective of this work was to develop pharmaceutical forms that pro-vide the local release of triamcinolone in the oral cavity. Mucoadhesive films and tablets were made from natural polymers such as gellan and pectin. The buccal films were prepared by sol-vent casting using different amounts of polymers. The raw materials and films were characte-rized physically chemically using vibrational spectroscopy (FTIR and Raman) and X-ray diffraction. The physical and mechanical properties of the films were also evaluated. In addi-tion, the mucoadhesive and drug dissolution tests were performed. The tablets were prepared by direct pressing with the natural polymers. Different parameters in relation to mixtures and formulations were evaluated such as the flow properties of the constituent powders, average weight, hardness, friability and disintegration. In relation to oral films, these were successfully obtained by a simple method, without the use of crosslinking agents, acids or organic solvents. All presented good results in the evaluated properties, however the formulations with interme-diate amounts of polymers were the best. Among the tablet formulations prepared, only 4 sho-wed good characteristics, however, the dissolution test results showed that these formulations have the ability to act as a controlled drug delivery system.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 126-143).

O tratamento farmacológico de patologias bucais é conduzido, geralmente, por via de adminis-tração local. No entanto, devido ao pouco tempo de permanência do fármaco no local de ação, esse tratamento pode ser bastante comprometido. Assim, este trabalho teve por objetivo o de-senvolvimento de formas farmacêuticas que proporcionem a liberação local de triancinolona na cavidade oral. Foram produzidos filmes e comprimidos mucoadesivos a partir de polímeros naturais como gelana e pectina. Os filmes bucais foram preparados por meio de evaporação do solvente (solvent casting) utilizando diferentes quantidades de polímeros. As matérias-primas e os filmes foram caracterizados fisico quimicamente utilizando espectroscopia vibracional (in-fravermelho com transformada de Fourier e Raman) e difração de raios X. As propiedades físicas e mecânicas dos filmes também foram avaliadas. Além disso, realizou-se os ensaios de mucoadesividade e de dissolução do fármaco. Os comprimidos foram preparados por com-pressão direta usando como base os polímeros naturais. Diferentes parâmetros em relação as misturas e as formulações foram avaliados tais como as propriedades de fluxo dos pós consti-tuintes, peso médio, dureza, friabilidade e desintegração. Em relação aos filmes bucais, estes foram obtidos com sucesso através de um método simples, sem a utilização de agentes reticu-lantes, ácidos ou solventes orgânicos. Todos apresentaram bons resultados nas propriedades avaliadas, no entanto as formulações com quantidades intermediarias de polímeros foram as melhores. Dentre as formulações de comprimidos preparadas, apenas 4 apresentaram boas ca-racterísticas, no entanto, os resultados dos ensaios de dissolução mostraram que estas formula-ções têm capacidade de agir como sistema de liberação controlada de fármacos. / Pharmacological treatment of oral pathologies is usually conducted by local administration. However, due to the short time the drug stays in the site of action, this treatment can be quite compromised. Thus, the objective of this work was to develop pharmaceutical forms that pro-vide the local release of triamcinolone in the oral cavity. Mucoadhesive films and tablets were made from natural polymers such as gellan and pectin. The buccal films were prepared by sol-vent casting using different amounts of polymers. The raw materials and films were characte-rized physically chemically using vibrational spectroscopy (FTIR and Raman) and X-ray diffraction. The physical and mechanical properties of the films were also evaluated. In addi-tion, the mucoadhesive and drug dissolution tests were performed. The tablets were prepared by direct pressing with the natural polymers. Different parameters in relation to mixtures and formulations were evaluated such as the flow properties of the constituent powders, average weight, hardness, friability and disintegration. In relation to oral films, these were successfully obtained by a simple method, without the use of crosslinking agents, acids or organic solvents. All presented good results in the evaluated properties, however the formulations with interme-diate amounts of polymers were the best. Among the tablet formulations prepared, only 4 sho-wed good characteristics, however, the dissolution test results showed that these formulations have the ability to act as a controlled drug delivery system.