O cuidado com as áreas doadoras de enxertos de pele merece constante atenção, visto que desencadeia desconforto por dor e restrição de movimentos. Existem evidências de que a estimulação elétrica pode acelerar a cicatrização de feridas e produzir analgesia, e que diferentes parâmetros físicos podem interferir nas respostas apresentadas. O objetivo do estudo foi avaliar o efeito da polaridade da corrente na cicatrização e na dor. Para tanto foram comparados os efeitos da estimulação elétrica de alta voltagem (EAV), polarizada, e a estimulação elétrica nervosa transcutânea (ENT), despolarizada, no tratamento das áreas doadoras de pacientes queimados. Para tanto, foram avaliados 48 voluntários do sexo masculino randomizados em três grupos: submetidos à estimulação elétrica de alta voltagem (GEAV), média idade de 34,2(±9,8) anos, n=17; submetidos à estimulação elétrica nervosa transcutânea (GENT), com 34(±9,5) anos, n= 16; e não submetido à estimulação elétrica ou grupo controle (GC) média de idade 35(±9,5 anos), n= 15. Os procedimentos terapêuticos foram aplicados nas extremidades da área doadora, no primeiro pós-operatório, até a epitelização completa. As variáveis avaliadas foram avaliação clínica, o tempo (dias) de epitelização, estimado pelo desprendimento do curativo primário sobre a lesão, avaliação da dor pela escala numérica de dor, a temperatura cutânea pela termografia infravermelha, qualidade da cicatriz (book de fotos, escala ® Vancouver, software Image J ). Após a análise dos dados, foi aplicado o teste de Shapiro-Wilk, em seguida o comportamento pré e pós-intervenção intragrupo foi aplicado o teste Wilcoxon. Para comparação entre os grupos foi efetuado teste de Kruskal-Wallis seguido de post-hoc de Dunn, em todos os casos foi utilizado o nível de significância de 5% (p<0,05). Os achados apontam que o tempo de desprendimento do curativo Rayon das áreas doadoras foi significativamente menor para GEAV apresentando (p<0,033). Houve redução significativa da dor (p<0,05) para o GEAV e para o GENT, quando comparado ao GC. A quantidade relacionada a solicitação de analgésicos foi reduzida para os grupos estimulados, com diferença significativa do GEAV versus GC (p<0,002) e GENT versus GC (p<0,001). Não houve diferença significativa na temperatura cutânea entre os grupos. Não houve diferença significativa no escore final da escala Vancouver e nem quantidade de crostas entre os grupos. A polaridade da corrente pode ter influenciado no tempo de epitelização, porém não interferiu na dor e na qualidade da área doadora. / The cares with donor areas of skin grafts deserve constant attention, since it triggers discomfort due to pain and movement restriction. There are evidences that electric stimulation may accelerate wound healing and produce pain relief. The objective of this study was to evaluate the effect of electric current polarity on the healing and pain. Therefore, the effects of stimulation on high voltage pulsed current (HVPC) and nervous transcutaneous stimulation (TENS) were compared in the treatment of donor areas of burns victims. Therefore, 48 volunteers of the male sex were randomized between three groups: submitted to high voltage pulsed current stimulation (GHVPC), with 34.2 (± 9.8) years, n=17; submitted to nervous transcutaneous stimulation (GTENS), with 34 (±9.5) years, n=16; and nonsubmitted to stimulation group, or control group (GC), average age of 35 (± 9,5) years, n=15. The therapeutic procedures were applied on the edges of the donor area, at the first postoperative, until complete healing. The evaluated variables were ephitelization time (days), estimated by the unfastening of the primary curative on the wound, evaluation of pain by numerical scale of pain, skin temperature by infrared thermography, scar quality (photo book, Vancouver scale and Image J® software). After data analysis, the Shapiro-Wilk test was applied, and the Wilcoxon test was applied to the before and after intervention. Kruskal-Wallis test followed by Dunn post-hoc was used to compare the groups. In all cases, the significance level of 5% (p<0.05) was used. The findings indicate that the time of release of the Rayon dressing from the donor sites was significantly reduced for GEAV (p <0.033). A reduction of pain relief was significant (p<0.05) for the GEAV and for the GENT, when compared to the GC. Amount the number of solitation for analgesic drugs was decreased for the groups stimulated with significant difference of the GEAV verse GC (p<0.002) and GENT verse GC (p <0.001). Change in cutaneous temperature was not significant between groups. There was no difference significant in score of Vancouver scale and in the number of crusts in the groups. The polarity of the current might have influenced the healing time, however not the pain nor the quality of the donor site.

Bacterial infections are a serious problem for patients with burns and other wounds. Such burn wound infection accounts for the pathogenic bacteria by colonising onto burned areas. Therefore, the need for detection and inhibition of such bacterial colonisation requires a methodology for sensing/killing pathogenic bacteria. This research project aims to design a ‗smart‘ wound dressing system which can respond to the microbiological environment of the wound via a simple colour change and will release antimicrobials only when required. Two strains of pathogenic bacteria Staphylococcus aureus (MSSA 476) and Pseudomonas aeruginosa (PAO1) were used in the study. The non-pathogenic bacterium E.coli (DH5α) was used as a control organism as it does not secrete virulence factors and therefore does not lyse membranes of vesicles. The key contributions of this thesis are outlined below. Firstly, an initial responsive nanocapsule system was studied. The fundamental work with giant unilamellar vesicles proved such a responsive system can provide antimicrobial properties when antimicrobial agents were encapsulated within the vesicles. Secondly, partially polymerised vesicles—polydiacetylene/phospholipid vesicles were then developed to improve vesicle stability. The vesicle system was optimised by varying molar concentration of diacetylene monomers (TCDA) in order to obtain relatively stable vesicles as well as sensitivity to the toxins secreted by the pathogenic strains. Measurements proved that the polydiacetylene/phospholipid vesicles can respond to pathogenic bacteria when fluorescent dye/antimcirobials were encapsulated in the vesicles. Finally, a simple prototype dressing was constructed. Plasma polymerised maleic anhydride (pp-MA) deposited onto non-woven polypropylene was shown to be a good method to stabilise vesicles via covalent bonding. Vesicle adhered to pp-MA non-woven polypropylene showed the ability to inhibit/kill the pathogenic strains, quantified by the Japanese Industry Standard assay and also gave a fluorimetric colour response in the presence of pathogenic bacteria when a fluorescent dye is encapsulated within vesicles. Other simple prototypes were also attempted by using hydrogels (gelatine and collagen) to maintain vesicle stability as well as promote tissue healing.

Orientador: Flávio Ruas de Moraes / Banca: Claudinei da Cruz / Banca: Mauricio Laterça Martins / Resumo: Neste trabalho foi avaliada a cinética da evolução do processo cicatricial em Piaractus mesopotamicus alimentados com ração suplementada por cromo trivalente e/ou parede celular de Saccharomyces cerevisiae, distribuídos ao acaso em quatro grupos; T1= controle sem suplemento; T2= com 0,3% de parede celular; T3= com 0,3% parede celular +18 mg de cromo trivalente/kg de ração; T4= com 18 mg de cromo trivalente/kg de ração. Após o período de alimentação de 105 dias, foram realizadas incisões na pele para a remoção da epiderme e derme. A avaliação do processo cicatricial foi realizada macroscopicamente e microscopicamente após um, três, sete, 14, 21, 28 e 35 dias de induzir as lesões. Na avaliação macroscópica foi utilizada a área de retração da ferida representativa da média de cada grupo. Para avaliação microscópica foram retirados fragmentos de pele do bordo superior até o inferior incluindo a musculatura. Os dados obtidos foram submetidos à análise de variância, e quando significativos foram comparados pelo teste de Tukey a 5% de probabilidade. No exame macroscópico e na análise histológica, as lesões dos animais que receberam ração suplementada por parede celular de S. cerevisiae ou cromo trivalente os eventos do processo cicatricial foram antecipados em relação ao grupo controle. A área de retração das feridas apresentou diferença entre os dias de avaliação demonstrando um aumento progressivo da velocidade do processo cicatricial, entretanto sem diferenças entre os grupos. A espessura da epiderme e derme, o número de células caliciformes e de neovasos não diferiram entre os grupos, contudo demonstraram a evolução do processo cicatricial nos diferentes tempos avaliados. / Abstract: This study evaluated the kinetics of the evolution of the healing process in P. mesopotamicus fed with ration supplemented with trivalent chromium and/or cell wall of S. cerevisiae, distributed at random into four groups; T1 = control without supplement, T2 = with 0.3% of the cell wall, T3 = with 0.3% cell wall +18 mg of trivalent chromium/kg diet, T4 = with 18 mg of trivalent chromium/kg diet. After the feeding period of 105 days, incisions were made in the skin to remove the epidermis and dermis. The evaluation of the healing process was performed macroscopically and microscopically after one, three, seven, 14, 21, 28 and 35 days induction of injuries. For macroscopical evaluation was used the rate of retraction of the wound representative of the average of each group. For microscopical evaluation pieces of skin were removed from the upper to the lower including muscle. Data were submitted to analysis of variance and when significant were compared by Tukey test at 5% probability. On macroscopic examination and histological analysis, the lesions of animals fed with ration supplemented with cell wall of S. cerevisiae or trivalent chromium the events of the healing process were anticipated in the control group. The area of wound retraction was different between the days of valuation showing a progressive increase of speed the healing process, however without differences between groups. The thickness of the epidermis and dermis, the number of goblet cells and neovascularization didn't differ between groups, although, demonstrated the evolution of the healing process in different moments. / Mestre

Orientador: Rosemary Adriana Chierici Marcantonio / Banca: Luís Carlos Spolidório / Banca: Letícia Theodoro / Resumo: O laser cirúrgico de Er,Cr:YSGG (érbio, impregnado por cromo: ítrio, scandio, gálio, garnet) tem sido utilizado no tratamento cirúrgico periodontal, embora poucos estudos histológicos sejam encontrados na literatura. O objetivo deste estudo foi comparar histologicamente a utilização do laser de Er-Cr: YSGG em tecido mole e ósseo em relação à técnicas cirúrgicas convencionais. Foram utilizados 20 ratos, divididos aleatoriamente em 3, 7, 15 e 30 dias, com 5 animais por período. Em cada animal foram realizados os procedimentos (P) nas regiões de dorso e calota, respectivamente: PI) Incisão no dorso com bisturi de lâmina no 15; PII) Incisão no dorso com Laser Er,Cr:YSGG (150mJ de energia, 2.0W de potência), PIII) Defeito ósseo na calota com fresa diamantada esférica em baixa rotação sob refrigeração; PIV) Defeito ósseo na calota com Laser Er,Cr:YSGG (150mJ de energia, 3.0W de potência). Os procedimentos foram randomicamente distribuídos entre lado direito e esquerdo de cada animal. A análise histológica foi avaliada de acordo com a intensidade da resposta inflamatória, presença de áreas necróticas e processo de regeneração tecidual. A análise histométrica dos defeitos ósseos foi confirmada pelo teste estatístico paramétrico ANOVA e complementado pelo teste Bonferroni. Para os procedimentos na região de dorso pode-se observar que nos períodos iniciais houve melhor resposta histológica para o PI, embora no período de 30 dias os procedimentos I e II apresentavam resultados semelhantes. A análise histométrica confirmou uma maior formação óssea na calota com o uso do laser de Er,Cr:YSGG aos 30 dias (79.96±10.30%) em relação ao procedimento com fresa aos 15 (62.05±4.84%) e 30 dias (58.23±9.99%). Na análise histológica em tecido duro, observou-se necrose óssea superficial... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The Er,Cr:YSGG (Erbium, Chromium:Yttrium-Scandium-Gallium-Garnet) surgical laser has been demonstrated as an useful instrument on dentistry since its wavelength (2,78μm) is strongly absorbed by water and hydroxyapatite. The aim of this study was to histologically compare the in vivo healing process that occurs in soft and hard tissues incisions made by Er,Cr:YSGG laser or conventional periodontal surgeries techniques. Twenty rats were used and divided into 4 groups of 5 different periods. On each animal was performed the following procedures (P) in the soft and hard tissue: PI) dorsum incision with scalpel number 15; PII) dorsum irradiation with 2.0W Er,Cr:YSGG laser; PIII) skull defect made by a diamond spherical bur with water refrigeration; PIV) skull defect created by 3.0W Er,Cr:YSGG laser. The procedures were randomly distributed into right and left side of each animal. After sacrifice 3, 7, 15 and 30 days post-surgery the histological examinations were performed. Histometric analysis of the bone defects was confirmed by the parametric statistic test ANOVA and complemented by the Bonferroni test. In the first periods, the histological findings in the dorsum were better in the PI group, although by day 30 PI and PII showed similar healing response. The procedures in the skull revealed superficial necrosis in all periods in the PIV group, although this group had much more evidence of bone formation at day 30 than PIII. The histometric analysis also confirmed a better tissue response for laser application in the skull than conventional techniques, with 79.96±10.30% bone formation by day 30 with Er,Cr:YSGG laser and 62.05±4.84% by day 15 and 58.23±9.99% by day 30 with bur. The utilization of Er,Cr:YSGG laser in soft tissue caused a minimal delay in the healing initial periods but the... (Complete abstract, click electronic access below) / Mestre

Orientador: Carlos Alberto Hussni / Coorientador: Carlos Roberto Teixeira / Banca: Marcos Jun Watanabe / Banca: Rogério Loesch Zacariotti / Resumo: As lesões cutâneas em répteis possuem diferentes causas e decorrem em cicatrizes e disecdise, predispondo a dermatites futuras, indesejáveis, podendo determinar a retirada do animal da exposição e levar ao abandono destes quando criados domesticamente. Com o objetivo de comparar diferentes produtos tópicos e procedimentos na busca da cicatrização da pele, foram utilizados doze lagartos adultos, machos, da espécie Tupinambis merianae. Em cada animal foram produzidas quatro lesões circulares na pele do dorso, dispostas em quadrilátero. Compostos três grupos de quatro animais, o Grupo 1 compreende o comparativo entre solução fisiológica e policresuleno; no Grupo 2, tintura de iodo a 2% e o não tratamento, considerado como controle; o Grupo 3 clorexidine e o enxerto autólogo de pele. As lesões do lado direito destinaram-se à análise macroscópica diária, com planimetria a cada sete dias e observação do tempo total de cicatrização. Das lesões esquerdas, foram coletadas amostras para biópsias e conseqüentes exames histopatológicos, a cada sete dias. Os lagartos apresentaram processo cicatricial semelhante aos mamíferos, notoriamente mais lento, entretanto. O enxerto não se mostrou vantajoso neste experimento, sofrendo necrose nos quatro animais. O policresuleno destacou-se por impermeabilizar a ferida em 4 dias, enquanto que os demais grupos obtiveram o mesmo efeito entre 8 e 12 dias. As lesões se mostraram completamente epitelizadas entre 40 e 53 dias, não havendo diferença entre os grupos quanto ao tempo total de cicatrização, retração tecidual e aparência da cicatriz / Abstract: Skin lesions in reptiles are caused by different factors and result in scarring and dysecdysis, predisposing to dermatitis. It may let the animal out of exhibitions or lead to the abandonment when created as pets. In order to compare different topical products and procedures for skin repair, twelve adult male tegu lizards (Tupinambis merianae) were used. Four circular square arranged skin lesions were created at the dorsum of each animal. Animals were divided in three groups (four animals/group) and each group was treated with two medications. In Group 1, animals were treated with saline and policresulen, in Group 2, with tincture of iodine and untreated (control group), and in Group 3, chlorhexidine and autologous skin graft. The lesions on the right side were macroscopicaly evaluated daily and the planimetry was realized every seven days. The left lesions were sampled every seven days for histopathological examination. The lizards showed healing process similar to mammals; however, it was notably slower. In this experiment, the skin graft showed no advantage, undergoing necrosis in all four animals. Policresulen closed the wounds in four days, while other groups had the same effect between 8 and 12 days. The lesions were completely epithelialized between 40 and 53 days, with no difference between groups regarding the total time of healing, tissue retraction and appearance of the scar / Mestre

Orientador: Alexandre Lima de Andrade / Coorientador: Suely Regina Mogami Bomfim / Banca: Paulo César Ciarlini / Banca: Regina Kiomi Takahira / Resumo:Biotécnicas como o plasma rico em plaquetas (PRP) vêm sendo amplamente utilizadas na reparação e regeneração teciduais, objetivando acelerar a cicatrização de feridas. O potencial do PRP para diminuir o tempo de recuperação dos traumas depende da concentração plaquetária, de sua função, e de seus fatores de crescimento. A escassez de protocolos para obtenção do PRP em cães, aliada às variações dos mesmos, dificulta o emprego desta biotécnica. O objetivo deste estudo foi testar três diferentes protocolos para obtenção do PRP e verificar sua eficácia na espécie canina. Foram colhidos 15 mL de sangue de 20 cães sadios, sendo as amostras submetidas aos protocolos para obtenção do PRP segundo Anitua, Sonnleitner e Vendramin. As concentrações médias de plaquetas proporcionadas por cada protocolo foram de 247%, 351% e 439%, respectivamente. O protocolo de Vendramin se sobressaiu em relação aos demais, com melhor concentração plaquetária no PRP, e apresentou boa reprodutibilidade / Abstract:Biotechnology as the platelet-rich plasma (PRP) have been widely used in tissue repair, aiming to accelerate the healing of wounds. The potencial of PRP to reduce the recovery time of injuries depends on the concentration of platelets, its function and their growth factors. The lack of protocols for obtaining PRP in dogs, combined with variations of the same, hinders the use of biotech. The aim of this study was to test three different protocols for obtaining PRP and verify effectiveness in dogs. 15 mL were collected blood of 20 healthy dogs, and the samples were subjected to protocols to obtain PRP second Anitua, Sonnleitner and Vendramin. The mean concentrations of each protocol provided by platelets were 247%, 351% and 439% respectively. The protocol Vendramin stood in relation to other, better platelet count in PRP, and has good reproducibility / Mestre

Orientador: Andrigo Barboza De Nardi / Banca: Mônica Vicky Bahr Arias / Banca: Geórgia Modé Magalhães / Resumo: A enxertia cutânea é uma técnica cirúrgica simples e bastante útil para o reparo de feridas, principalmente nas quais existe dificuldade da aplicação do fechamento primário, ou de outras técnicas reconstrutivas. Entretanto, para a sobrevivência do enxerto, é necessário que o leito da ferida esteja saudável e com a presença de um tecido de granulação exuberante. O objetivo deste trabalho foi avaliar a aplicação da membrana amniótica e da laserterapia como potenciais estimulantes da cicatrização em enxertos aplicados em feridas sem tecido de granulação. Para isso, foram utilizados 42 coelhos divididos em quatro grupos de tratamento, grupo controle (GC), grupo membrana (GM), grupo laser (GL) e grupo membrana e laser (GML), submetidos a avaliações macro e microscópicas dos enxertos. Na avaliação macroscópica foi possível notar que os pacientes dos grupos onde a membrana amniótica foi utilizada com o intuito de estimular a reepitelização (GM e GML) apresentaram acentuada reação inflamatória, falha de integração do enxerto e consequente necrose do mesmo. Já os pacientes do grupo GL apresentaram melhor aspecto do enxerto no último dia de avaliação. Na análise microscópica, observou-se intensa integração do enxerto à derme, reepitelização acentuada e escassas células inflamatórias no local do enxerto nos pacientes do grupo GL. O oposto foi observado nos pacientes dos grupos GM e GML, onde aparentemente a presença da membrana amniótica induziu resposta inflamatória bastante consistente na região adjacente à membrana e ao enxerto, resultando em rejeição da membrana. Por último, a formação de colágeno não se correlaciona com outros fatores como inflamação e necrose, em nenhum dos grupos de tratamento. Desta forma, é possível afirmar que a laserterapia mostrou ser efetiva, contribuindo para o processo cicatricial e integração do enxerto. Já a membrana amniótica canina não deve ser... / Abstract: Skin grafting is a simple surgical technique and useful to repair wounds, especially those where there is a difficulty to apply primary closure skin or other reconstructive techniques. However, for the graft survival it is necessary that the healthy wound bed and the presence of an exuberant granulation tissue. This study aimed to evaluate the application of amniotic membrane and laser therapy as potential stimulating healing in grafts applied in wounds without granulation tissue. For this, we used 42 rabbits divided into four treatment groups, control group (CG), membrane group (MG), laser group (LG) and membrane and laser group (MLG), submitted to macroscopic and microscopic evaluation. The macroscopic examination was possible to note that the patients of the groups where the amniotic membrane was used in order to stimulate re-epithelialization (MG and MLG) presented evidences associated with severe inflammatory reaction, graft integration failure and consequent necrosis. In LG patients apparently had the best aspect of the graft in the last valuation date. On the microscopic examination, there was intense integration of the graft to the dermis, high reepithelialization level and scarce inflammatory cells in the graft site of LG group patients. The opposite was observed in patients in the MG and MLG groups, where apparently the presence of amniotic membrane seems to have induced very consistent inflammatory response in the region adjacent to the membrane and the graft, evincing that there was a rejection of the membrane. Finally, the collagen formation does not correlated with other factors such as inflammation and necrosis in any of the treatment groups. We can conclude that laser therapy was effective, contributing to the healing process and integration of the graft. Thus, the canine amniotic membrane should not be used for this purpose because it causes intense inflammatory reaction besides to avoid graft nutrition / Mestre

Peroxissome proliferator-activated receptor-alfa (PPAR-?) é um fator de transcrição nuclear envolvido na regulação do metabolismo de lipídeos e da inflamação. PPAR? pode estar relacionado com a modulação da cicatrização de feridas cutâneas, que é um processo multifatorial, dependente de mecanismos de sinalização celular e de inflamação. Deste modo, o objetivo deste trabalho foi analisar o papel do receptor PPAR? na cicatrização de feridas cutâneas induzidas experimentalmente e a sua relação com o metabolismo sistêmico após tratamento com agonista do PPAR?. Para tanto, foram realizadas feridas na pele da região dorsal de camundongos 129/SvEv, que foram tratados diariamente com o agonista de PPAR?, Gemfibrozil, por via oral ou tópica. Os animais foram acompanhados durante 240h pós-cirúrgico (p.c.) para a análise do reparo cutâneo e alterações metabólicas que poderiam ser induzidas pela ativação de PPAR?. Os camundongos tratados apresentaram melhor cicatrização após ativação de PPAR? com 100 ou 50 mg/kg/dia de agonista por via oral ou tópica, respectivamente. O tratamento oral induziu cicatrização mais rápida somente após 24h, 48h e 72h p.c., enquanto que os animais tratados com Gemfibrozil tópico apresentaram cicatrização mais precoce em todos os tempos avaliados. A indução de feridas alterou o metabolismo sistêmico dos camundongos que demonstraram significativa perda de peso e redução de triglicérides, independentemente do tratamento. Porém, a ativação de PPAR? não alterou a glicemia ou a função hepática. Na análise histopatológica das feridas foi verificado infiltrado inflamatório, composto principalmente por neutrófilos e outras células polimorfonucleares. Entretanto, o tratamento com Gemfibrozil tópico levou a um menor infiltrado inflamatório e diferenciada deposição de colágeno após 10 dias p.c. Além disso, houve diminuição do acúmulo de neutrófilos, macrófagos e eosinófilos quando comparados aos animais que receberam apenas o veículo. O tratamento tópico promoveu menor acúmulo de linfócitos TCD4+, TCD8+ e T??, e ainda diferenciado influxo de células dendríticas para a lesão. No entanto, não houve diferença em relação a células T reguladoras nos linfonodos drenantes, mas os animais tratados apresentaram diminuição de Foxp3 nas células CD4+CD25-. Em conclusão, PPAR? atua no reparo cutâneo, e sua ativação local acelera a cicatrização por meio da modulação da inflamação na pele. Finalmente, os resultados sugerem que PPAR? pode ser alvo importante para novas terapias que visam melhorar a cicatrização de feridas, especialmente quando ativado no local da lesão. / Peroxissome proliferator-activated receptor alpha (PPAR?) is a nuclear transcription factor involved in the regulation of lipid metabolism and inflammation. PPAR? may be associated to the modulation of wound healing, which is a multifactorial process dependent on mechanisms of cell signaling and inflammation. Then this work aimed to analyze the role of PPAR? receptor in experimental cutaneous wound healing and its relationship to the systemic metabolism of mice treated with a PPAR? agonist. For this, skin wounds were performed in the dorsal region of 129/SvEv mice, treated daily with the PPAR? agonist, Gemfibrozil, by oral or topical route. Mice were followed for 240h post-surgery (p.s.) for skin repair and metabolic changes that could be induced by PPAR? activation. There was improved wound healing in mice treated with 100 or 50 mg/Kg of PPAR? agonist by oral or topical route respectively. The oral treatment induced a better repair in the early 24h, 48h and 72h p.s. while mice treated by topical application of Gemfibrozil presented faster healing in all times evaluated. Wound\'s induction affected the systemic metabolism of mice leading to significant weight loss. PPAR? agonist did not alter glucose, triglycerides or liver function, although all injured animals had a significant decrease on triglycerides levels in the early times p.s., independent on the treatment. In histopathological examination of the wounds it was observed inflammatory infiltrate, composed mainly of neutrophils and other polymorphonuclear cells. However, topical treatment with PPAR? agonist led to lower inflammatory infiltrate and differentiated collagen deposition 10 days p.s. Furthermore, there was decrease of neutrophil, macrophages and eosinophils influx when compared to untreated mice. Topical treatment led to decrease in the TCD4+, TCD8+ e T?? lymphocytes accumulation in the lesions, and differentiated dendritic cell influx to the wounds. However there was no difference regarding CD4+CD25+ T cells in lymph nodes, but treated mice showed decrease Foxp3 expression. In conclusion, the triglycerides serum level was altered in the course of wound healing and may be associated to skin lesion, while PPAR? agonist acts in wound repair by accelerating healing and modulating neutrophil influx to the skin. Finally, our results suggested that PPAR? may be an important target for novel therapies aimed at improved wound healing, especially when administered topically.

INTRODUCTION: The cornea forms the anterior-most barrier of the eye, consisting of a non-keratinized pseudostratified squamous epithelium, a collagen-based stroma, and an endothelium. It is completely avascular, yet the most densely innervated structure in the human body. The sensory nerves project from the ophthalmic branch of the trigeminal cranial nerve into the limbal/stromal interface. From there, the nerves branch and ascend into Bowman’s membrane, a basal lamina delineating the epithelium from the stroma, and project into the epithelium as free nerve endings. Injury to the corneal epithelium can potentially lead to impaired vision if the wound healing process is not properly initiated. Immediately after injury, nucleotides such as ATP are released and bind to purinergic receptors known to be located in epithelial cell membranes, thereby initiating epithelial cell migration to close the wound. Malfunctions in the interactions between the corneal nerves and their epithelial counterparts during the wound healing process are thought to contribute to the attenuated wound healing characteristic of diabetes. However, the precise nature of these interactions, how they facilitate wound healing, and how they are impaired in diabetes, is not well understood. OBJECTIVES: Previously, our lab has shown that a member of purinergic family receptors (P2X7) is localized in the basal epithelial cells and becomes relocated to the leading edge of the wound after injury. When the relocation is inhibited, migration is attenuated. Additionally, it is known that diabetic mouse models display slower wound healing rates. The present study has three aims: (1) to replicate the characteristic sub-basal whorl organization of the corneal nerves in organ-cultured corneas; (2) to elucidate the connections between patterns of corneal innervation and purinergic receptor expression; and (3) to understand how these patterns interact to facilitate normal wound healing and how these interactions are disrupted in a diabetic model. METHODS: Our approach was to use immunohistochemistry of dissected mouse and to visualize the tissue using confocal microscopy. Sensory innervation profiles from diet induced obesity (DIO) mouse corneas and their wildtype C57Bl6 counterparts were compared in unwounded and wounded tissue. To image the nerves a methanol fixation protocol was optimized to examine the sub-basal plexus and the apical nerves. Corneas were dissected, stained with beta III-tubulin, which identifies nerves, and with an antibody to the P2X7 purinergic receptor, which is expressed in the epithelium and nerves. Trephine induced epithelial abrasion injuries were made on separate DIO and control models to compare re-epithelialization and re-innervation between the diseased and healthy states. Corneas were imaged using a Zeiss LSM 700 laser scanning confocal microscope and optical images were taken through the cornea over a distance averaging 115 microns. Corneas were imaged using a macro tiling plugin, stitching 3x3 optical z-stacks into composite images. The 3x3 tiles were created to image the central whorl, as well as the peripheral nerve fibers. Co-localization of P2X7 and betaIII tubulin were determined by thresholding using ImageJ/FIJI software. RESULTS: The elegant organization of the centralized sub-basal whorl of the control mouse was disrupted in the DIO mouse cornea, appearing fragmented and incomplete. Analysis of 7.5 and 15 wk corneas showed the whorl to be present at 7.5 wks. Average apical nerve fiber projection length was decreased in DIO cornea. Yet, analyses at each epithelial layer demonstrated overall increased apical nerve density in the DIO corneas as compared to control while sub-basal nerve density decreased dramatically. Stromal nerves remained equivalent. P2X7 did co-localize to the large stromal nerve fibers but it was difficult to show the localization along the sub-basal nerve plexus. However in cross-section images, P2X7 displayed an intracellular polarity, and was present along the apical surface of the columnar basal epithelial cells lining the basement membrane. This localization may suggest the presence of P2X7 expressing sensory nerves, which may be ideally poised for communication with the basal cells after injury. CONCLUSIONS: These data support the hypothesis that there is indeed a difference between diabetic and control corneal innervation. While wound healing differences due to the interaction between sensory nerves and the localization of P2X7 in epithelium at the leading edge remain to be fully elucidated, the novel finding of P2X7 expression in corneal nerves confirms a potential role of purinergic receptor and nerve coordination in conducting the wound healing response.

Wound repair is a complex series of events that begins immediately after wounding, and can continue for a number of months to years. Various physiological and mechanical factors may impair the healing response, resulting in a chronic wound, characterised by a sustained inflammatory response. One of the main cells involved in both the inflammatory phase and proliferation phase of wound healing is the macrophage. There are thought to be different activation states which allow the macrophage to be involved in the two different phases of wound healing, namely the classically activated macrophage and the alternatively activated macrophage. Changes in the number of classically activated/alternatively activated macrophages in the wound is likely to have an effect on wound healing. Therefore a more thorough understanding of macrophage activation states during wound healing would broaden the understanding of the role of this cell in this process. The overall aim of this project was to investigate whether diabetic bone marrow progenitor cells or macrophages respond to activation stimuli differently in comparison to wild type cells. The hypothesis of this thesis is that diabetic macrophages will not respond to appropriate stimuli and thus alternative and classical macrophages will not behave 'appropriately', resulting in impaired healing. The results of this thesis indicate that impaired wound healing in the diabetic environment may be due to both the diabetic wound environment itself and intrinsic differences in diabetic macrophages. This work indicates that signals from the wound environment activates and influences macrophages, as these cells do not express activation markers until they enter the wound environment. However, the culture system devised in this study indicates that even before activation with signals they would receive in the wound environment, diabetic cells are more pro-inflammatory and have impaired migration. In addition, these macrophages respond differently to activation supporting the hypothesis that the macrophages are intrinsically different and that diabetic cells do not behave 'appropriately' which could contribute to impaired wound healing.