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.

The regulated differentiation and polarisation of macrophages are essential for successful wound healing process. During wound repair, macrophages are involved in the early inflammatory process of healing, as well in later regenerative phases by producing cytokines and growth factors relevant for each stage. Their plasticity made macrophages able to change their phenotype from M1 inflammatory during the inflammatory phase of healing to M2 reparative during regenerative phases of healing. Diabetes affects the ability of macrophages to mature from the bone marrow and on their ability to polarise to different phenotypic subsets. Whereas the non-diabetic macrophages can mature normally to M2 macrophages during mid-stages of healing, diabetic wound continues o display immature proinflammatory macrophages resulting in mixed M1/M2 macrophages in the wound that remain until late stages of healing. We previously showed that sustained expression of Hoxa3 reduced the-the excessive number of leukocytes recruited to the wound, suggesting an anti-inflammatory effect of Hoxa3 upon all leukocytes population. Hoxa3 protein transduction also promoted the differentiation of HSC/P into pro-angiogenic Gr1+CD11b+ myeloid cells. Here we showed that Hoxa3 promoted the differentiation of macrophages and upregulated the transcriptional machinery controlling macrophage differentiation, in THP-1 monocytes and primary macrophages from non-diabetic and diabetic mice. Using qRT-PCR and protein analysis of bone marrow derived macrophages from diabetic mice, we showed that Hoxa3 upregulated the master regulator of macrophages differentiation, Pu.1 transcriptionally and post- transcriptionally and that Hoxa3 protein interacted with Pu.1 protein in vitro and in vivo within macrophages proposing a mechanism of their regulation. Hoxa3 also inhibited proinflammatory markers in classically activated macrophages and augmented pro-healing markers in alternatively activated macrophages. Investigating the IL-4/Stat6 pathway of M2 macrophage activation revealed that Hoxa3 upregulated Stat6 and increased Stat6 phosphorylation, a novel effect of Hoxa3 on the signaling pathway of alternative macrophage activation. In vivo analysis of Hoxa3's effect on wound derived macrophages in diabetic mice, confirmed that Hoxa3 promoted the generation of pro-healing macrophages and showed reduced Nos2+ (M1) cells and increased Arg1+ (M2) cells suggesting that Hoxa3 can rescue the phenotype of diabetic macrophages in the wound. Altogether, this work has delineated the specific role of Hoxa3 in rescuing maturation and phenotype of diabetic macrophages thereby providing a better understanding of the therapeutic role of this transcription factor for myeloid cells dysregulation in diabetes.

Thesis (M.S.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 1982. / MICROFICHE COPY AVAILABLE IN ARCHIVES AND ENGINEERING / Includes bibliographical references. / by Elizabeth Hsiu-Yun Chen. / M.S.

Orientador: Valdir Gouveia Garcia / Resumo: O objetivo do estudo foi avaliar, histológicamente e biometricamente, a influência da associação de diferentes comprimentos de onda de lasers em baixa intensidade, sobre o processo de reparo em feridas cutâneas abertas provocadas no dorso de ratos. Foram utilizados 72 ratos, os quais foram divididos em 3 grupos com 24 animais cada, sendo: Grupo I - controle (sem tratamento); Grupo II (tratamento com laser 685 nm) e grupo III (tratamento com laser 685 seguido do laser 780 nm na mesma sessão). O laser foi utilizado de modo contínuo e em contato de forma pontual, ponteiras de 50 mW, durante 80 s, nas feridas do grupo II e 40 s (laser 685 nm ) mais 40 segundos (laser 780 nm) nas do grupo III, totalizando 4 J de energia e densidade energética de 200 J/cm2 em ambos os grupos. O diâmetro das feridas foram medidas em 0 h, 3, 7 e 14 d de pós-operatório e, os dados foram submetidos à análise estatística. Nos intervalos de 3, 7 e 14 d, 8 animais de cada grupo foram sacrificados e as peças foram submetidas a processamento histológico e coradas pela técnica da hematoxilina e eosina e tricrômico de Massom. Concluiu-se: 1) Grupo II e III evidenciaram resultados histológicos mais diferenciados que Grupo I; 2) Grupo III demonstrou reparação mais diferenciada que o Grupo I e o Grupo II; 3) do ponto de vista biométrico, o maior grau de contração foi observado nas feridas do Grupo III, seguido das do grupo II e I, no período de 14 dias. / Abstract: This investigation aimed to study, histologically, the influence of association of different wave-length of low intensity laser on the healing process in cutaneous wound provoked at the dorsum of rats. Seventy-two rats, 3 groups of 24 animals each, were used, namely: Group I - control (without treatment); Group II (treatment with laser of 685 nm) and Group III (treatment with laser of 685 nm associated with laser of 780 nm in the same session). In Group II, the laser was applied continuously with a point of contact of 50 mW during 80 s. In Group III, the laser of 685 nm was applied and, followed with laser of 780 nm for 40 s in each application. A total of 4 J of energy and 200 J/cm2 of energetic density were applied in both groups. The diameter of the wound was measured at baseline, 3, 7 and 14 post-operative days and the results were submitted to statistical analysis. At intervals of 3, 7 and 14 days, 8 animals of each group were sacrificed and the pieces were submitted to histological processing and were stained by hematoxiline and eosine and Massom's tricromic techniques. It was concluded that: 1) Group II and III showed histological results statistically different from Group I; 2) Group III demonstrated a statistically differentiated healing in comparison to Group I and II; 3) at the point of view of biometrics, the greatest degree of contraction was observed in Group III, followed by Group II and I, at the period of 14 days. / Mestre

"July, 2001" / Addendum inserted in back. / Includes bibliographical references (leaves 250-284) / xxiii, 284 leaves : ill. (some col.), plates (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Surgery, 2001

<p>Syftet med denna litteraturstudie var att via litteraturen beskriva nutritionens betydelse för sårläkning. Studien genomfördes som en deskriptiv litteraturstudie där metoden bestod av att söka vetenskapliga artiklar i databaserna Medline, Cinahl och Academic Search Elite. Sjutton artiklar valdes ut. Resultatet visade att patienter med bensår eller trycksår i många fall inte når upp till de näringsrekommendationer som finns. Det visade sig vara vanligt att dessa patienter ligger vid gränsen för att vara undernärda. Det upptäcktes att det är sällan som kostintaget täcker energibehovet. Framför allt var zinknivåerna genomgående väldigt låga och det visar även att det är brist på en hel del andra ämnen. Ämnen som visat sig speciellt fördelaktiga för sårläkning är protein, vitamin C, zink samt arginin. För patienter med brännskador däremot har det visat sig att saltet ornithine α-ketoglutarate minskar proteinkatabolismen samt förbättrar proteinsyntesen, vilket är en förutsättning för sårläkning. Slutsatsen med föreliggande studie var att nutritionen är viktig och kan ha en avgörande betydelse för sårläkning.</p> / <p>The purpose with this literature study was that through literature describe the nutrition’s significance for healing wounds. The study was made as a descriptive literature study where the method was conducted by searching scientific articles in the databases Medline, Cinahl and Academic Search Elite. Seventeen articles were chosen. The result showed that patients with leg ulcers or pressure sores in many cases don’t attain the recommendations for nutrition that are available. It showed that it is normal that these patients are on the border of being undernourished. It was discovered that the nutrient intake rarely covers the energy needs. Above all the zinc levels were throughout very low and that shows that there is also lack of some other substances. The substances that have been shown especially good for healing wounds are protein, vitamin C, zinc and arginine. However, for patients with burn injuries the salt ornithine α-ketoglutarate has been shown to reduce protein catabolism and also improve protein synthesis, which are essential to healing wounds. The conclusion from this study was that nutrition may be an important but also a determining factor to healing wounds.</p>

Syftet med denna litteraturstudie var att via litteraturen beskriva nutritionens betydelse för sårläkning. Studien genomfördes som en deskriptiv litteraturstudie där metoden bestod av att söka vetenskapliga artiklar i databaserna Medline, Cinahl och Academic Search Elite. Sjutton artiklar valdes ut. Resultatet visade att patienter med bensår eller trycksår i många fall inte når upp till de näringsrekommendationer som finns. Det visade sig vara vanligt att dessa patienter ligger vid gränsen för att vara undernärda. Det upptäcktes att det är sällan som kostintaget täcker energibehovet. Framför allt var zinknivåerna genomgående väldigt låga och det visar även att det är brist på en hel del andra ämnen. Ämnen som visat sig speciellt fördelaktiga för sårläkning är protein, vitamin C, zink samt arginin. För patienter med brännskador däremot har det visat sig att saltet ornithine α-ketoglutarate minskar proteinkatabolismen samt förbättrar proteinsyntesen, vilket är en förutsättning för sårläkning. Slutsatsen med föreliggande studie var att nutritionen är viktig och kan ha en avgörande betydelse för sårläkning. / The purpose with this literature study was that through literature describe the nutrition’s significance for healing wounds. The study was made as a descriptive literature study where the method was conducted by searching scientific articles in the databases Medline, Cinahl and Academic Search Elite. Seventeen articles were chosen. The result showed that patients with leg ulcers or pressure sores in many cases don’t attain the recommendations for nutrition that are available. It showed that it is normal that these patients are on the border of being undernourished. It was discovered that the nutrient intake rarely covers the energy needs. Above all the zinc levels were throughout very low and that shows that there is also lack of some other substances. The substances that have been shown especially good for healing wounds are protein, vitamin C, zinc and arginine. However, for patients with burn injuries the salt ornithine α-ketoglutarate has been shown to reduce protein catabolism and also improve protein synthesis, which are essential to healing wounds. The conclusion from this study was that nutrition may be an important but also a determining factor to healing wounds.

The plasminogen activator (PA) system has been shown to be intimately involved in wound healing. However, the role of this system in the initiation and resolution of inflammation during healing process remained to be determined. The aims of this thesis were to investigate the molecular mechanism underlying the interaction between the PA system and the inflammatory system during wound healing and to explore the therapeutic potential of plasminogen in various wound-healing models. The role of plasminogen in the inflammatory phase of the healing process of acute and diabetic wounds was studied first. Our data showed that administration of additional plasminogen to wild-type mice accelerates the healing of acute wounds. After injury, both endogenous and exogenous plasminogen are bound to inflammatory cells and are transported to the wound site, which leads to activation of inflammatory cells. In diabetic db/db mice, wound-specific accumulation of plasminogen does not take place and the inflammatory response is impaired. However, when additional plasminogen is injected, plasminogen accumulates in the wound, the inflammatory response is enhanced, the signal transduction cascade is activated and the healing rate is significantly increased. These results indicate that administration of plasminogen may be a novel therapeutic strategy to treat different types of wounds, especially chronic wounds in diabetes. The role of plasminogen at the later stage of wound healing was also studied in plasminogen-deficient mice. Our data showed that even if re-epithelialization is achieved in these mice, a prolonged inflammatory phase with abundant neutrophil accumulation and persistent fibrin deposition is observed at the wound site. These results indicate that plasminogen is also essential for the later phases of wound healing by clearing fibrin and resolving inflammation. The functional role of two physiological PAs during wound healing was further studied in a tympanic membrane (TM) wound-healing model. Our data showed that the healing process was clearly delayed in urokinase-type PA (uPA)-deficient mice but not in tissue-type PA (tPA)-deficient mice. Less pronounced keratinocyte migration, abundant neutrophil accumulation and persistent fibrin deposition were observed in uPA-deficient mice. These results indicate that uPA plays a central role in the generation of plasmin during the healing of TM perforations. Finally the therapeutic potential of plasminogen in the TM wound-healing model was studied. Our data showed that local injection of plasminogen restores the ability to heal TM perforations in plasminogen-deficient mice in a dose-dependent manner. Plasminogen supplementation also potentiates healing of acute TM perforations in wild-type mice, independent of the administration method used. A single local injection of plasminogen in plasminogen-deficient mice can initiate healing of chronic TM perforations resulting in a closed TM with a continuous but rather thick outer keratinocyte layer. Three plasminogen injections lead to a completely healed TM with a thin keratinizing squamous epithelium covering a connective tissue layer that can start to reorganize and further mature to its normal appearance. In conclusion, our results suggest that plasminogen is a promising drug candidate for the treatment of chronic TM perforations in humans.  Taken together, our data indicate that plasminogen is a novel inflammatory regulator that promotes wound healing.