Perfusion and Ultrasonication Produce a Decellularized Porcine Whole-Ovary Scaffold with a Preserved Microarchitecture.

The application of decellularized scaffolds for artificial tissue reconstruction has been an approach with great therapeutic potential in regenerative medicine. Recently, biomimetic ovarian tissue reconstruction was proposed to reestablish ovarian endocrine functions. Despite many decellularization methods proposed, there is no established protocol for whole ovaries by detergent perfusion that is able to preserve tissue macro and microstructure with higher efficiency. This generated biomaterial may have the potential to be applied for other purposes beyond reproduction and be translated to other areas in the tissue engineering field. Therefore, this study aimed to establish and standardize a protocol for porcine ovaries' decellularization based on detergent perfusion and ultrasonication to obtain functional whole-ovary scaffolds. For that, porcine ovaries (n = 5) were perfused with detergents (0.5% SDS and 1% Triton X-100) and submitted to an ultrasonication bath to produce acellular scaffolds. The decellularization efficiency was evaluated by DAPI staining and total genomic DNA quantification. ECM morphological evaluation was performed by histological, immunohistochemistry, and ultrastructural analyses. ECM physico-chemical composition was evaluated using FTIR and Raman spectroscopy. A cytocompatibility and cell adhesion assay using murine fibroblasts was performed. Results showed that the proposed method was able to remove cellular components efficiently. There was no significant ECM component loss in relation to native tissue, and the scaffolds were cytocompatible and allowed cell attachment. In conclusion, the proposed decellularization protocol produced whole-ovaries scaffolds with preserved ECM composition and great potential for application in tissue engineering.

Alpha-tocopherol-loaded polycaprolactone nanoparticles improve the inflammation and systemic oxidative stress of arthritic rats.

Background and aim: The present study investigated the effects of orally administered α-tocopherol-loaded polycaprolactone nanoparticles on the articular inflammation and systemic oxidative status of middle-aged Holtzman rats with Freund's adjuvant-induced polyarthritis, a model for rheumatoid arthritis. Intraperitoneally administered free α-tocopherol provided the reference for comparison. Experimental procedure: Two protocols of treatment were followed: intraperitoneal administration of free α-tocopherol (100 mg/kg i.p.) or oral administration of free and nanoencapsulated α-tocopherol (100 mg/kg p.o.). Animals were treated during 18 days after arthritis induction. Results: Free (i.p.) and encapsulated α-tocopherol decreased the hind paws edema, the leukocytes infiltration into femorotibial joints and the mRNA expression of pro-inflammatory cytokines in the tibial anterior muscle of arthritic rats, but the encapsulated compound was more effective. Free (i.p.) and encapsulated α-tocopherol decreased the high levels of reactive oxygen species in the brain and liver, but only the encapsulated compound decreased the levels of protein carbonyl groups in these organs. Both free (i.p.) and encapsulated α-tocopherol increased the α-tocopherol levels and the ratio of reduced to oxidized glutathione in these organs. Conclusion: Both intraperitoneally administered free α-tocopherol and orally administered encapsulated α-tocopherol effectively improved inflammation and systemic oxidative stress in middle-aged arthritic rats. However, the encapsulated form should be preferred because the oral administration route does not be linked to the evident discomfort that is caused in general by injectable medicaments. Consequently, α-tocopherol-loaded polycaprolactone nanoparticles may be a promising adjuvant to the most current approaches aiming at rheumatoid arthritis therapy.

Dynamics of the natural genesis of ß-TCP/HAp phases in postnatal fishbones towards gold standard biocomposites for bone regeneration.

The search for gold-standard materials for bone regeneration is still a challenge in reconstruction surgery. The ratio between hydroxyapatite (HAp) and ß-tricalcium phosphate (ß-TCP) in biphasic calcium phosphate ceramics (BCPs) is one of the most important factors in osteoinduction promotion and controlled biodegradability, configurating what is currently considered as a possible gold standard material for bone substitution in reconstructive surgery. Exploring the natural genesis of the HAp and ß-TCP phases in fishbones during their postnatal growth, this study developed a biphasic bioceramic obtained from the calcination of Nile tilapia (Oreochromis niloticus) bones as a function of their ages. The natural genesis dynamics of the structural evolution of the ß-TCP and HAp phases were characterized by physicochemical methods, taking into account of the age of the fish and the material processing conditions. Thermal analysis (TGA / DTA) showed complete removal of the organic matter and transitions associated with the transformation of carbonated hydroxyapatite (CDHA) to HAp and ß-TCP phases. After calcination at 900 °C, the material was characterized by: X-ray diffraction (XRD) and refinement by the Rietveld method; Fourier Transform Infrared Spectroscopy with Attenuated Total Reflection (FTIR-ATR); Raman spectroscopy; Scanning Electron Microscopy (SEM) and Flame Atomic Absorption Spectroscopy (FAAS). The analysis allowed identification and quantitative estimate of the variations of the HAp and ß-TCP phases in the formation of the BCPs. The results showed that the decrease in ß-TCP against the increase in the HAp phases is symmetrical to the dynamics of the natural genesis of these phases, surprisingly maintaining the balanced phase proportion even when bones of young fishes were used. The microstructure analysis confirms the observed transformation. In addition, in vivo tests demonstrated the osteoinductive potential of BCP scaffolds implanted in an ectopic site, and their remarkable regenerative functionality, as bone graft, was demonstrated in alveolar bone after tooth extraction. MTT cytotoxicity assay for BCP samples for MC3T3-E1 pre-osteoblasts and L929 fibroblasts cells showed viability equal or higher than 100%. A logistic empirical model is presented to explain the three stages of HAp natural formation with fish age and it is also compared to the fish size evolution.

Bioperformance Studies of Biphasic Calcium Phosphate Scaffolds Extracted from Fish Bones Impregnated with Free Curcumin and Complexed with ß-Cyclodextrin in Bone Regeneration.

Fish bones are a natural calcium phosphate (CaP) sources used in biomaterials production for bone regeneration. CaP scaffolds can be enriched with other substances with biological activity to improve bone repair. This study aimed to evaluate the physicochemical properties and bone regeneration potential of biphasic calcium phosphate (BCP) scaffolds impregnated with free curcumin (BCP-CL) or complexed with ß-cyclodextrin (BCP-CD) compared to BCP scaffolds. Rietveld's refinement showed that BCP is composed of 57.2% of HAp and 42.8% of ß-TCP and the molar ratio of Ca/P corresponds to 1.59. The scaffolds presented porosity (macro and microporosity) of 57.21%. Apatite formation occurred on the BCP, BCP-CL, and BCP-CD surface, in vitro, in SBF. Micro-Raman technique showed a reduction in the dissolution rate of ß-TCP in the curcumin-impregnated scaffolds over time, and in vivo studies on critical-size defects, in rat calvaria, had no additional regenerative effect of BCP-CL and BCP-CD scaffolds, compared to BCP scaffolds. Despite this, the study showed that curcumin impregnation in BCP scaffolds prolongs the release of the ß-TCP phase, the BCP- phase with the higher osteoinductive potential, representing an advantage in tissue engineering.

In vivo evaluation of interactions between biphasic calcium phosphate (BCP)-niobium pentoxide (Nb2O5) nanocomposite and tissues using a rat critical-size calvarial defect model.

Natural or synthetic biomaterials are increasingly being used to support bone tissue repair or substitution. The combination of natural calcium phosphates with biocompatible alloys is an important route towards the development of new biomaterials with bioperformance and mechanical responses to mimic those of human bones. This article evaluated the structural, physical, mechanical and biological properties of a new mechanical improved nanocomposite elaborated by association of fish biphasic calcium phosphate (BCP) and niobium pentoxide (Nb2O5). The nanocomposite (Nb-BCP) and the pure BCP, used as a positive control, were obtained by powder metallurgy. The density, porosity and microhardness were measured. The structural analysis was determined by X-ray diffraction (XRD) and the biological properties were studied in histological sections of critical size calvaria defects in rats, 7, 15, 30, 45 and 60 days after implantation of disks of both materials. Morphological description was made after scanning electron microscopy (SEM) and optical microscopy analysis. After sintering, the Nb-BCP nanocomposite presented four crystalline phases: 34.36% calcium niobate (CaNb2O6), 21.68% phosphorus niobium oxide (PNb9O25), 42.55% ß-tricalcium phosphate (Ca3(PO4)2) and 1.31% of niobium pentoxide (Nb2O5) and exhibited increases of 17% in density, 66% in Vickers microhardness and 180% in compressive strength compared to pure BCP. In vivo study, showed biocompatibility, bioactivity and osteoconductivity similar to pure BCP. SEM showed the formation of globular accretions over the implanted nanocomposites, representing one of the stages of bone mineralization. In conclusion, the BCP and Nb2O5 formed a nanocomposite exhibiting characteristics that are desirable for a biomaterial, such as bioperformance, higher ß-TCP percentage and improved physical and mechanical properties compared to pure BCP. These characteristics demonstrate the promise of this material for supporting bone regeneration.

Resistance exercise training induces subcutaneous and visceral adipose tissue browning in Swiss mice.

Aerobic exercise training (AER) may promote several adaptations in white adipose tissue (WAT), including a phenotypic change known as browning. The present study aimed at assessing if resistance exercise training (RES) would be as efficient as AER in inducing a brown-like adipocyte reprogramming in WAT. Thirty Swiss male mice were randomly divided into 3 groups with 10 animals each: 1) sedentary (SED), 2) AER, and 3) RES. After the adaptation training, an incremental test was performed at the beginning of each week to adjust training load. Mice were submitted to 8 wk of AER or RES. After the experimental period, inguinal and retroperitoneal WAT (iWAT and rpWAT) and brown adipose tissue (BAT) were collected. The prescription of AER and RES was effective in increasing the performance of both groups. Also, RES presented a lower body weight than AER/SED. AER and RES reduced the area of iWAT and rpWAT adipocytes and the lipid area of BAT, induced an increase of vascular endothelial growth factor (VEGF) and cluster of differentiation 31 (CD31) and uncoupling protein 1 (UCP-1), and increased the expression of selective genes of brown and beige phenotype in adipocytes after 8 wk. In general, we demonstrated here that AER and RES training similarly induced the browning of iWAT and rpWAT.NEW & NOTEWORTHY Aerobic exercise training (AER) induces the browning of white adipose tissue, turning adipocytes multilocular, highly vascularized and expressing uncoupling protein 1 (UCP-1). The current study compared the efficiency of resistance to aerobic exercise training to promote a brown-like phenotype. Our results suggest that both types of training similarly induce subcutaneous and visceral adipose tissue browning.

Immune response and Raman scattering assessment in rats skin after contact with Fusarium oxysporum metabolites.

The secondary metabolites produced by Fusarium can cause disease and death when consumed and produce biological responses even in the absence of the microorganism. The IL-6, TNF-α and TGF-ß1 cytokines immune reactivity was associated with histopathological and physico-chemical changes in skin of immune competent rats after administration of Fusarium oxysporum crude extract. Rats were intradermally injected with 50 µl of 0.5 mg/ml crude extract and were euthanized at 3, 6, 12 and 24 h after injection. The inflammatory response was quantified by enzyme myeloperoxidase activity and by immunohistochemical method to detect the IL-6, TNF-α and TGF-ß1. Physico-chemical analysis was performed using FT-Raman Spectroscopy. The inflammatory response was most intense at 6 and 12 h after crude extract administration and the most significant histopathological changes were observed in the dermis. Myeloperoxidase activity was intense from 3 to 24 h after injection. The immunostaining of pro-inflammatory cytokines IL-6 and TNF-α peaked at 6 h. Immunostaining for TGF-ß1 was highest at 12 and 24 h. FT-Raman spectral analysis showed both, the most intense Fusarium interaction with the skin at 6 h, as revealed by the changes in the stretching of -CH bands (3100-2800 cm-1) in the dermis, and skin recovery trending after 12 h after crude extract injection. The results showed that secondary metabolites stimulated histopathologic changes and inflammatory responses even in the absence of the fungus, increasing myeloperoxidase activity and pro-inflammatory cytokine expression besides promoting physico-chemical changes.

Analysis of bone formation and membrane resorption in guided bone regeneration using deproteinized bovine bone mineral versus calcium sulfate.

Guided Bone Regeneration (GBR) is a technique based on the use of a physical barrier that isolates the region of bone regeneration from adjacent tissues. The objective of this study was to compare GBR, adopting a critical-size defect model in rat calvaria and using collagen membrane separately combined with two filling materials, each having different resorption rates. A circular defect 8 mm in diameter was made in the calvaria of Wistar rats. The defects were then filled with calcium sulfate (CaS group) or deproteinized bovine bone mineral (DBBM group) and covered by resorbable collagen membrane. The animals were killed 15, 30, 45 and 60 days after the surgical procedure. Samples were collected, fixed in 4% paraformaldehyde and processed for paraffin embedding. The resultant sections were stained with H&E for histological and histomorphometric study. For the histomorphometric study, the area of membrane was quantified along with the amount of bone formed in the region of the membrane. Calcium sulfate was reabsorbed more rapidly compared to DBBM. The CaS group had the highest percentages of remaining membrane at 15, 30, 45 and 60 days, compared to the DBBM group. The DBBM group had the highest amount of new bone at 45 and 60 days compared to the CaS group. Based on these results, it was concluded that the type of filling material may influence both the resorption of collagen membrane and amount of bone formed.

Exocellular extract of Fusarium oxysporum, fungus free, is able to permeate and act selectively in skin.

The skin is an important gateway for Fusarium infection in humans. Our hypothesis is that metabolites produced by Fusarium oxysporum should change the barrier structure to permeate the skin. Male Wistar rats received a topical application of a solution (0.05 mg/mL) of Fusarium metabolites. The animals were euthanized 3, 6, 12, 24 h after and the skin was processed for immunostaining by laminin and E-cadherin to investigate whether the Fusarium metabolites can break the barrier of healthy skin. Other techniques were employed: H&E to study the morphology; metalloproteinase-9 (MMP-9), TUNEL, and PCNA immunostaining to evaluate the inflammation, cell death, and proliferation, respectively. There was an inflammatory response mainly centered in the dermis. Qualitatively, the skin of the experimental group showed reduced E-cadherin and laminin immunostaining at 3, 12, and 24 h. Higher intensity staining by TUNEL at 3 h, and PCNA at 6, 12, and 24 h. There was intense MMP-9 activity at 6, 12, and 24 h. None of analyses revealed any changes in the epidermis. It was concluded that the fraction was able to permeate the skin and act selectively in dermis, inducing inflammatory response, increasing MMP-9 immunostaining, inducing apoptosis, and reducing E-cadherin and laminin immunostaining.

Combination of Histopathology and FT-Raman Spectroscopy for the Study of Experimental Paracoccidioidomycosis in the Spleen.

Paracoccidioides brasiliensis (Pb) can disseminate through the lymphatic and hematogenic pathways. As a result the spleen and other lymphoid organs are targets of paracoccidioidomycosis. There are few studies describing this disease in the spleen and more detailed descriptions are required. This study combines Histopathology and Fourier Transform Raman Spectroscopy (FT-Raman) methods to study spleen infected by Pb. The Swiss mice were euthanized after 1, 2, 4 and 8 weeks of infection with Pb, and their spleens were removed for ex vivo analyzes. Histopathological evaluation revealed that the red pulp was the area most affected, presenting the highest concentration of yeasts, inflammatory cells and cells in apoptosis, with no observation of typical granuloma formation. The main physical-chemical changes were detected in the spectral ranges between 1730-1590 cm-1 and 1390-1280 cm-1 attributed to amide I and vibrational deformation mode of CH3 and CH2 molecules, respectively. The amide I was correlated to total protein content and CH3 and CH2 molecules to collagen of the spleen. The association between histological and physical-chemical methods enabled detection of several alterations in the spleen, including apoptosis, contributing to a better understanding of paracoccidioidomycosis, a tropical neglected disease.

Radiation used for head and neck cancer increases virulence in Candida tropicalis isolated from a cancer patient.

BACKGROUND: Studies have shown that radiation from radiotherapy increases the yeast colonization of patients. However it is not clear, if such radiation alters the yeast itself. The aim of the present study was therefore to report the direct impact of gamma radiation on Candida tropicalis. METHODS: C. tropicalis was obtained from a patient with a carcinoma, a suspension of this yeast containing 2.0 × 103 colony forming units per milliliter was prepared. It was submitted to gamma radiation dosage similar to that used in the treatment of head and neck cancer. After a cumulative dose of 7200 cGy some virulence attributes of C. tropicalis, including macro and micromorphological characteristics, adhesion and biofilm abilities, murine experimental infection and phagocytosis resistance were evaluated on irradiated and non-irradiated yeasts. RESULTS: After irradiation the colony morphology of the yeast was altered from a ring format to a smooth appearance in most colonies. Scanning electron microscopy revealed notable differences in the structures of both these colonies and the yeast cells, with the loss of pseudohyphae following irradiation and an increase in extracellular matrix production. The adherence and biofilm production of the yeast was greater following irradiation, both in terms of the number of yeasts and total biomass production on several abiotic surfaces and TR146 cells. The phagocytic index of the irradiated yeasts was not statistically different; however, the presence of cellular debris was detected in the kidneys of infected animals. Mice infected with irradiated yeasts developed an infection at the site of the yeast inoculation, although systemic infection was unchanged. CONCLUSIONS: Our findings show for the first time that C. tropicalis, one of the most important yeasts from colonization, which cause fatal candidemia in cancer patients, is affected by gamma irradiation, with changes to its virulence profile.

γ-irradiation from radiotherapy improves the virulence potential of Candida tropicalis.

AIM: To evaluate if radiation used in radiotherapy can cause changes in the virulence potential of Candida tropicalis ATCC 750. MATERIALS & METHODS: C. tropicalis was exposed in vitro to identical dose and scheme of irradiation would be used in patients with head and neck cancer. Some virulence parameters were analyzed before and after irradiation. RESULTS: Colony morphologies were irreversibly affected by irradiation. Increase in growth rate, filamentation, adhesion on cell lines and phagocytosis process were also observed. Overall the irradiated C. tropicalis cells became more efficient at causing systemic infection in mice. CONCLUSION: γ-radiation induced important changes in C. tropicalis increasing its virulence profile, which could directly affect the relationship between yeasts and hosts.

Insulin complexed with cyclodextrins stimulates epithelialization and neovascularization of skin wound healing in rats.

INTRODUCTION: Skin lesions are a significant public health problem, above all that wounds fail to heal properly and become chronic. Due to its reepithelization action, insulin has the potential to heal skin lesions, by stimulating the proliferation and migration of keratinocytes, angiogenic stimulus, and increasing collagen deposition. In the present study insulin was complexed with 2-hydroxypropyl-ß-cyclodextrin (HPßCD) and its wound healing effect and inclusion complex (HPßCD-I) were evaluated in excisional wounds in the skin of rats. MATERIAL AND METHODS: Three different gel based pharmaceutical forms were created: carbopol 940® base gel, an insulin gel comprising the base gel plus 50 IU of insulin and a gel complex comprising the base gel plus (HPßCD) complexed with insulin (HPßCD-I) were used to verify wound healing in vitro and in vivo assays. RESULTS: The wounds in the skin of rats were treated with gel containing HPßCD-I not cytoxically irritating and cytotoxic. Analysis of cell proliferation and measurement of the length and thickness of the epidermis showed that HPßCD-I prolonged the proliferation and migration of keratinocytes. Revascularization analysis of lesions treated with HPßCD-I compared to those treated with insulin found that angiogenic stimulus was less intense, but more constant and prolonged in the modified release process. There was increased deposition of type I and III collagen fibers in accordance with the treatment time. CONCLUSION: Therefore, the slow release of complexed insulin modulated the reepithelialization process by stimulating cell proliferation and migration of keratinocytes, favoring greater concentration of serum insulin, modulating inflammatory response, matrix remodeling and promoting neovascularization. Angiogenesis extended by the steady release of insulin can be effective in the treatment of chronic wounds.

Increased of the hepatocytes and splenocytes apoptosis accompanies clinical improvement and higher survival in mice infected with Trypanosoma cruzi and treated with highly diluted Lycopodium clavatum.

Recent evidence includes apoptosis as a defense against Trypanosoma cruzi infection, which promotes an immune response in the host induced by T cells, type 1, 2 and 17. Currently, there is no medicine completely preventing the progression of this disease. We investigated the immunological and apoptotic effects, morbidity and survival of mice infected with T. cruzi and treated with dynamized homeopathic compounds 13c: Kalium causticum (GCaus), Conium maculatum, (GCon), Lycopodium clavatum (GLy) and 7% alcohol solution (control, vehicle compounds, GCI). There was significant difference in the increase of apoptosis in the treated groups, compared with GCI, which might indicate action of the compounds in these cells. Infected animals treated with Lycopodium clavatum presented better performance compared with other groups. GLy showed a higher amount of hepatocytes and splenocytes undergoing apoptosis, higher number of apoptotic bodies in the liver, predominance of Th1 response, increased TNF-α and decreased IL-6, higher survival, lower morbidity, higher water consumption, body temperature, tendency to higher feed intake and weight gain compared with GCI. Conium maculatum had worse results with increased Th2 response with increased IL-4, worsening of the infection with early mortality of the animals. Together, these data suggest that highly diluted medicines modulate the immune response and apoptosis, affecting the morbidity of animals infected with a highly virulent strain of T. cruzi, being able to minimize the course of infection, providing more alternative approaches in the treatment of Chagas disease.

Anethole reduces inflammation and joint damage in rats with adjuvant-induced arthritis.

AIM: This study evaluated whether anethole attenuates the inflammatory response and joint damage in a model of adjuvant-induced arthritis (AIA) in rats. METHODS: The animals were treated with 62.5-, 125-, or 250-mg/kg anethole daily for 21 days after AIA and necropsied on days 14 and 21 to evaluate the number of serum and synovial leukocytes (total and differential), serum cytokines (IL-2, IL-6, IL-12, IL-17, and TNF-α), and nitric oxide concentrations. Morphologic changes in the cartilage and bone of the femorotibial articulation in both left paw and right paw were studied in hematoxylin/eosin and Sirius Red-hematoxylin sections. RESULTS: Different doses of anethole suppressed paw swelling and the number of serum and synovial leukocytes. However, 250 mg/kg of anethole more effectively controlled local and systemic inflammation. Histological evaluation revealed significant prevention of cartilage damage and inflammatory infiltrate scores. Morphometric analysis showed pannus formation, the thickness of the articular cartilage, and bone resorption lower in the anethole-treated AIA group compared to untreated AIA group on both days 14 and 21. These significant anti-inflammatory effects in the anethole-treated AIA group were associated with downregulation of cytokines and nitric oxide levels. CONCLUSION: Therefore, anethole may be a useful intervention to treat inflammatory arthritis.

Employing photoacoustic spectroscopy in the evaluation of the skin permeation profile of emulsion containing antioxidant phenolic-rich extract of Melochia arenosa.

CONTEXT: Oxidative stress is an important factor modulating skin alterations. Melochia arenosa Benth. (Malvaceae) is a Brazilian plant with antimicrobial activity and antioxidant potential. OBJECTIVE: The objective of this study is to develop a topical formulation containing antioxidant phenolic-rich extract of M. arenosa and to evaluate its skin permeation profile. MATERIALS AND METHODS: Response surface methodology was used to maximize the total phenolic (TP) content of the extract and its antioxidant activity was evaluated by 2,2-diphenyl-1-picryl-hydrazyl (DPPH), 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and respiratory burst methods. An emulsion containing 1% optimized extract (OE) was developed and employed photoacoustic spectroscopy (PAS) for the determination of its skin permeation profile. The morphology of the skin was studied in histological sections stained with hematoxylin-eosin. RESULTS AND DISCUSSION: The optimum conditions predicted for the major extractive efficiency of the phenolics with 100% ethanol led extraction time 101 h and plant:solvent proportion 1:13.5 (w/v). OE presented TP = 724.6 ± 8.2 mg GAE/g extract and scavenging capacity of DPPH (IC50 value = 11.43 ± 0.14 µg/mL) and ABTS radicals (IC50 value = 35.42 ± 0.48 µg/mL). The production of ROS by neutrophils after stimulation with phorbol miristate acetate was lower when the OE was present in the reaction medium, endorsing its high antioxidant capacity. The data obtained by PAS indicated that the OE present in the emulsion has permeated and was distributed in the whole skin. No histopathological alterations were observed in the histological analysis. CONCLUSION: The formulation developed is a promising tool for skin care and could prevent the damage caused by oxidative stress.

Topical and Intradermal Efficacy of Photodynamic Therapy with Methylene Blue and Light-Emitting Diode in the Treatment of Cutaneous Leishmaniasis Caused by Leishmania braziliensis.

INTRODUCTION: The topical and intradermal photodynamic therapy (PDT) effect of methylene blue (MB) using light-emitting diode (LED) as light source (MB/LED-PDT) in the treatment of lesions of American cutaneous leishmaniasis (ACL) caused by Leishmania braziliensis in hamsters were investigated. METHODS: Hamsters were infected in the footpad with 4×10(7) promastigotes of L. braziliensis and divided in 4 groups: Control group was not treated, AmB group was treated with amphotericin B, MB-Id group received intradermal MB at the edge of the lesion and MB-Tp group received MB topic. After treatment with MB, the animals were illuminated using red LEDs at the 655 nm wavelength for 1 hour. The MB/LED-PDT was carried out three times a week for 12 weeks. RESULTS: Animals of MB-Tp group presented lesion healing with significant diminution in extent of the lesion, and reduced parasite burden compared to control group; however, no significant difference was seen compared to the AmB group. MB-Tp group also showed reconstitution of the epithelium, the formation of collagen fibers, organization in the epidermis, a little disorganization and inflammation in the dermis. MB-Id was ineffective in all parameters evaluated, and it was comparable to the control group results. CONCLUSION: These data show that PDT with the use of MB-Tp and LED may be an alternative for the treatment of ACL. However, additional studies are being conducted to assess the potential of MB/LED-PDT, alone or in combination with conventional therapy, for the treatment of ACL.

Histomorphometry of the organic matrix of the femur in ovariectomized rats treated with sodium alendronate.

OBJECTIVE: To evaluate the effect of two different concentrations of sodium alendronate on the quantity of organic matrix in the femur of rats with estrogen suppression caused by ovariectomy. METHODS: Sixty-days-old Wistar rats (Rattus norvegicus) were subjected to bilateral laparotomy to remove the ovaries. The animals were divided into a control group, in which they only underwent laparotomy; an ovariectomized group (OVX); an ovariectomized group treated with 1 mg/kg of alendronate (OVX 1 mg); and an ovariectomized group treated with 2 mg/kg of alendronate (OVX 2 mg). The rats received alendronate twice a week for 90 days. The left femur was then removed, fixed and processed for embedding in paraffin. Semi-serial sections stained with hematoxylin and eosin were used to determine the area occupied by organic bone matrix, by means of image analysis software. The animals' weights were obtained at the beginning and end of the experiment. RESULTS: The ovariectomized animals and those treated with 1 mg/kg of alendronate presented significant increases in body weight (p < 0.05), in comparison with the control group. Histomorphometric analysis revealed that in the animals treated with 2 mg/kg of alendronate, the area (µm(2)) occupied by organic matrix (1,81,900 ± 18,130) was similar (p > 0.05) to that of the non-ovariectomized control animals (2,04,800 ± 9590), which indicates that this medication had a preventive effect with regard to bone mass loss. CONCLUSION: The higher concentration of the medication, administered twice a week for 90 days, was more effective than the dose of 1 mg/kg over the same period.

OBJETIVO: Avaliar a efeito de duas concentrações diferentes de alendronato de sódio (ALN) sobre a quantidade de matriz orgânica no fêmur de ratas com supressão estrogênica provocada por ovariectomia. MÉTODOS: Ratas Wistar (Rattus norvegicus) com 60 dias foram submetidas a laparotomia bilateral para remoção dos ovários. Os animais foram divididos em grupo controle, no qual os animais foram apenas laparotomizados; grupo ovariectomizado (OVX); grupo ovariectomizado tratado com 1 mg/kg de ALN (OVX 1 mg); e grupo ovariectomizado tratado com 2 mg/kg de ALN (OVX 2 mg). Receberam ALN duas vezes por semana durante 90 dias. O fêmur esquerdo foi coletado, fixado e processado para inclusão em parafina. Cortes semisseriados corados com H&E foram usados para a obtenção, com um software de análise de imagens, da área ocupada por matriz óssea orgânica. O peso dos animais foi obtido no inicio e no fim do experimento. RESULTADOS: Os animais ovariectomizados e aqueles tratados com 1 mg/kg de ALN tiveram um aumento significativo (p < 0,05) no peso corporal quando comparados com o grupo controle. A análise histomorfométrica revelou que nos animais tratados com 2 mg/kg de ALN a área (µm2) ocupada por matriz orgânica (181.900 ± 18.130) foi semelhante (p > 0,05) àquela dos animais controle não ovariectomizados (204.800 ± 9.590), o que indica um efeito preventivo desse medicamento sobre a perda de massa óssea. CONCLUSÃO: A maior concentração do medicamento administrado duas vezes por semana por 90 dias foi mais eficaz do que a dose de 1 mg/kg no mesmo período.

Activity of synthetic chalcones in hamsters experimentally infected with Leishmania (Viannia) braziliensis.

The purpose of this study is to evaluate the toxicity of synthetic chalcones 1 and 2 in uninfected hamsters and anti-Leishmania activity of synthetic chalcones 1 and 2 in hamsters infected with Leishmania (Viannia) braziliensis. For the toxicity test, uninfected animals were treated with chalcones 1 and 2, and clinical and biochemical parameters and histological aspects of the liver and kidneys were assessed. Chalcones 1 and 2 were then intraperitoneally or topically administered (10 mg/kg body weight) three times per week in animals infected with promastigotes of L. (V.) braziliensis. We monitored the thickness of the infected footpads, determined parasitic load, performed histological analysis, and detected apoptosis in situ. The results were analyzed using Student's t test and Mann-Whitney test at a significance level of 5%. Neither of the chalcones showed toxicity. Chalcone 2 administered intraperitoneally significantly reduced the thickness of the infected footpad compared with the beginning of treatment. The parasite load of the lymph node and spleen was reduced in the groups treated with chalcones 1 (topical) and 2 (intraperitoneal). Chalcone 2 (topical) reduced parasite burden only in the lymph node. The histological analysis revealed reconstitution of the tissue and reductions of inflammation and apoptosis in the infected footpad in these groups. The synthetic chalcones 1 (topical) and 2 (intraperitoneal and topical) at a dose of 10 mg/kg showed anti-Leishmania activity in vivo, no renal or hepatic toxicity, and a reduction of apoptosis of the cells in the lesions. These chalcones may have substantial potential for the treatment of cutaneous leishmaniasis.

Nectandra falcifolia: potential phytopharmaceutical for skin damage protection designed by statistical approach and characterized by photoacoustic spectroscopy

AbstractPhytopharmaceutical products are being used in the treatment and prevention of health problems. Nowadays, the development and evaluation of novel pharmaceutical products is expensive and time consuming. A statistical approach is a good tool for optimal development processes. Nectandra falcifolia (Nees) J.A. Castigl. ex Mart. Crov. & Piccinini, Lauraceae, a Brazilian species, is reported as anti-inflammatory, anti-leishmanial and anti-microbial. However, there is little known about its chemical composition. For other species of Nectandra genus, the presence of antioxidant compounds is reported. In order to optimize the process of obtaining extract with high antioxidant activity, different extraction conditions were tested following a statistical approach. Two sequential experimental designs were used – first, a factorial 23 design, followed by central composite 22. The extracts manufactured by these experimental statistical matrixes had their antioxidant activity and phenolic contents quantified and the response surface plots were fitted in quadratic models and they predicted the best extraction condition for the best antioxidant activity. This standardized extract and its antioxidant activity were better evaluated by two complementary tests (ABTS and Burst respiratory). A topical formulation containing 1% (w/w) of standardized extract was prepared and used for an in vivo skin permeation study using a two-dose application. The photoacoustic spectroscopy was used to analyze the samples from the permeation study and the composition profile of standardized extract. In rat skin samples, the data demonstrated that for the higher dose of topical formulation (5 g/cm2), the standardized extract could cross skin and be seen in epidermis and dermis. This was not the case for the lower dose (2 g/cm2) which was only present in the epidermis. This information suggests that this novel standardized extract of N. falcifoliacould be explored for skin damage prevention or treatment for diseases developed by oxidative damage.