Intradermal Application of Crotamine Induces Inflammatory and Immunological Changes In Vivo

Crotamine is a single-chain polypeptide with cell-penetrating properties, which is considered a promising molecule for clinical use. Nevertheless, its biosafety data are still scarce. Herein, we assessed the in vivo proinflammatory properties of crotamine, including its local effect and systemic serum parameters. Sixty male Wistar rats were intradermically injected with 200, 400 and 800 µg crotamine and analyzed after 1, 3 and 7 days. Local effect of crotamine was assessed by determination of MPO and NAG activities, NO levels and angiogenesis. Systemic inflammatory response was assessed by determination of IL-10, TNF-α, CRP, NO, TBARS and SH groups. Crotamine induced macrophages and neutrophils chemotaxis as evidenced by the upregulation of both NAG (0.5–0.6 OD/mg) and MPO (0.1–0.2 OD/mg) activities, on the first and third day of analysis, respectively. High levels of NO were observed for all concentrations and time-points. Moreover, 800 μg crotamine resulted in serum NO (64.7 μM) and local tissue NO (58.5 μM) levels higher or equivalent to those recorded for their respective histamine controls (55.7 μM and 59.0 μM). Crotamine also induced a significant angiogenic response compared to histamine. Systemically, crotamine induced a progressive increase in serum CRP levels up to the third day of analysis (22.4–45.8 mg/mL), which was significantly greater than control values. Crotamine (400 μg) also caused an increase in serum TNF-α, in the first day of analysis (1095.4 pg/mL), however a significant increase in IL-10 (122.2 pg/mL) was also recorded for the same time-point, suggesting the induction of an anti-inflammatory effect. Finally, crotamine changed the systemic redox state by inducing gradual increase in serum levels of TBARS (1.0–1.8 μM/mL) and decrease in SH levels (124.7–19.5 μM/mL) throughout the experimental period of analysis. In summary, rats intradermally injected with crotamine presented local and systemic acute inflammatory responses similarly to histamine, which limits crotamine therapeutic use on its original form

Experimental models of malnutrition and its effect on skin trophism

FUNDAMENTOS: A pele, para exercer suas funções, necessita de níveis adequados de nutrientes. OBJETIVO: Analisar o trofismo cutâneo de ratos nutridos e desnutridos por meio de dois modelos de desnutrição. MÉTODOS: No Modelo Marasmo, utilizaram-se 60 ratos Wistar em controle dietético, dos quais 30 foram selecionados aleatoriamente para receber metade da dieta diária durante 60 dias. No Modelo Gelatina, empregaram-se 60 ratos, dos quais 30 receberam dieta associada a proteína de baixa qualidade (gelatina) durante 30 dias. Avaliou-se o estado nutricional dos animais por meio da massa corporal, dos sinais clínicos e da dosagem de albumina sérica. Após o período de desnutrição, fez-se a histologia da pele dos animais para análise da espessura da derme e epiderme com o software Leica Application Suite; nas lâminas coradas com tricrômio de Gomori, analisou-se a colagênese com o software ImageJ. RESULTADOS: A massa corporal dos animais desnutridos pelo marasmo e gelatina foi significativamente menor (p<0,0001 e p<0,0001) do que a dos grupos nutridos. Quanto à albumina sérica, não houve diferença entre os grupos nos dois modelos. Em relação à análise histológica da espessura da pele, os desnutridos apresentaram a derme significativamente menos espessa em comparação aos nutridos (p<0,0001 e p<0,0001). No que respeita à colagênese, os grupos desnutridos apresentaram menores percentuais de colágeno em relação aos nutridos (p<0,0005 e p<0,003). CONCLUSÕES: Os animais desnutridos pelos dois modelos apresentaram diminuição na espessura dérmica, confirmada histologicamente pelo menor percentual de colágeno, mostrando a influência negativa da desnutrição no trofismo cutâneoBACKGROUND: The skin requires adequate levels of nutrients to function properly. OBJECTIVE: To analyze skin trophism in well-nourished and undernourished rats using two models of malnutrition. METHODS: In the marasmus model, 60 Wistar rats were kept on a controlled diet, 30 being randomly selected to receive half the established diet for 60 days. In the gelatin model, 60 rats were used, 30 of which received a diet consisting of poor quality protein (gelatin) for 30 days. The nutritional status of the animals was evaluated according to body mass index, clinical signs and serum albumin measurement. After the period of malnutrition, histology was performed on the animals' skin to analyze the thickness of the dermis and epidermis using the Leica Application Suite software. Collagen was analyzed on slides stained with Gömöri trichrome using the ImageJ software program. RESULTS: The body mass index of the malnourished animals in the marasmus and gelatin groups was significantly lower than that of the well-nourished animals in the two groups (p<0.0001 in both models). With respect to serum albumin, there was no difference between the groups in either of the two models. In relation to the histological analysis of skin thickness, the dermis of the malnourished animals was significantly thinner compared to that of the well-nourished animals (p<0.0001 in both models). The percentage of collagen was lower in the malnourished animals compared to the well-nourished animals (p<0.0005 and p<0.003 in the marasmus and gelatin model, respectively). CONCLUSIONS: Skin thickness measurements were lower in the malnourished animals in both models, and this finding was histologically confirmed by the lower percentage of collagen, showing the negative effect of malnutrition on skin trophis

Evolution of Periodontal Disease: Immune Response and RANK/RANKL/OPG System

<div><p>Abstract The aim of this study was to evaluate markers of bone loss and immune response present in evolution of periodontal disease. One hundred and two Wistar rats were divided into three animals groups: PD0, without ligation and PD15 days and PD60 days, submitted to ligation placement with a sterile 3-0 silk cord in the cervical region of the upper first molar on both sides. Samples were obtained from the gingival tissue for histomorphometric analysis, immunohistochemical analysis of RANK, RANKL, OPG, characterization of the inflammatory infiltrate, quantification of nitric oxide, MCP-1, RANTES, IP10 chemokines, and expression of the TGF-b1, VEG, and bFGF. The number of inflammatory cells in gingival tissue was higher in PD60 samples. The collagen content and the area occupied by birefringent collagen fibers were lower for PD60. Differential leukocyte counting showed that there was a significantly higher polymorphonuclear influx in group PD15, while PD60 showed a greater number of lymphocytes. PD60 showed higher RANTES, IP-10, MCP-1 gene transcripts, as well as a higher nitric oxide concentration. Clinical evaluation revealed that the PD60 group presented an increase in furcal area. In conclusion, in this animal model the increase of RANK/RANKL and HGF markers is related to a specific immune response, and probably contributed to the evolution of periodontal disease. Investigating the effect of these biomarkers can help in targeted therapy for bone resorption, since blocking these can inhibit bone loss.</p></div

The vegetal biomembrane in the healing of chronic venous ulcers Biomembrana vegetal na cicatrização de úlceras venosas crônicas

BACKGROUND: The vegetal biomembrane has been used to treat cutaneous ulcers. OBJECTIVES: To assess the role of the vegetal biomembrane on the chronic venous ulcers treatment compared to treatment with collagenase cream. METHODS: Fourteen patients were selected to be treated with vegetal biomembrane and 7 with Fibrase®(CONTROL), followed clinically and photographically by the Wound Healing Index by ImageJ during 120 days and biopsied on the 1st and 30th days for histological examination. RESULTS: The vegetal biomembrane was better in promoting healing of the ulcers, especially on the inflammatory phase, confirmed by abundant exudation and wound debridement than the CONTROL group, on the 30th day. There was a greater tendency to angiogenesis followed by re-epithelialization with highest wound healing index on the 90th and 120th days. CONCLUSION: A combined analysis of clinical and histopathological findings suggests that the vegetal biomembrane acted as a factor inducing wound healing, especially on the inflammatory phase, confirmed by abundant exudation of the lesions promoting the transformation of the microenvironment of the chronic venous ulcers, and also stimulating angiogenesis and subsequent re-epithelialization.<br>FUNDAMENTOS: A biomembrana vegetal tem sido usada para tratamento de úlceras cutâneas. OBJETIVOS: Avaliar a ação da biomembrana vegetal no tratamento de úlceras venosas crônicas, comparando-a ao tratamento à base de colagenase. MÉTODOS: Foram selecionados 14 pacientes tratados com biomembrana vegetal e sete com Fibrase® (grupo controle), acompanhados clínico-fotograficamente pelo índice de cicatrização das úlceras (ICU) por 120 dias, por meio do software ImageJ, e biopsiados no primeiro e 30º dias para estudo histopatológico. RESULTADOS: A biomembrana vegetal foi superior em relação ao controle na cicatrização das úlceras no 30º dia, especialmente na fase inflamatória, confirmada pela exsudação abundante e pelo desbridamento. Houve tendência superior à angiogênese seguida de reepitelização com maiores ICUs no 90º e 120º dias. CONCLUSÃO: A análise conjunta dos achados clínicos e histopatológicos sugere que a biomembrana vegetal atuou como um fator indutor da cicatrização, especialmente na fase inflamatória, confirmada pela exsudação abundante das lesões, promovendo a transformação do microambiente das úlceras venosas crônicas e estimulando a angiogênese e a posterior reepitelização