In Vivo Assessment of Clobetasol Propionate-Loaded Lecithin-Chitosan Nanoparticles for Skin Delivery.

The aim of this work was to assess in vivo the anti-inflammatory efficacy and tolerability of clobetasol propionate (CP) loaded lecithin/chitosan nanoparticles incorporated into chitosan gel for topical application (CP 0.005%). As a comparison, a commercial cream (CP 0.05% w/w), and a sodium deoxycholate gel (CP 0.05% w/w) were also evaluated. Lecithin/chitosan nanoparticles were prepared by self-assembling of the components obtained by direct injection of soybean lecithin alcoholic solution containing CP into chitosan aqueous solution. Nanoparticles obtained had a particle size around 250 nm, narrow distribution (polydispersity index below 0.2) and positive surface charge, provided by a superficial layer of the cationic polymer. The nanoparticle suspension was then loaded into a chitosan gel, to obtain a final CP concentration of 0.005%. The anti-inflammatory activity was evaluated using carrageenan-induced hind paw edema test on Wistar rats, the effect of formulations on the barrier property of the stratum corneum were determined using transepidermal water loss measurements (TEWL) and histological analysis was performed to evaluate the possible presence of morphological changes. The results obtained indicate that nanoparticle-in-gel formulation produced significantly higher edema inhibition compared to other formulations tested, although it contained ten times less CP. TEWL measurements also revealed that all formulations have no significant disturbance on the barrier function of skin. Furthermore, histological analysis of rat abdominal skin did not show morphological tissue changes nor cell infiltration signs after application of the formulations. Taken together, the present data show that the use of lecithin/chitosan nanoparticles in chitosan gel as a drug carrier significantly improves the risk-benefit ratio as compared with sodium-deoxycholate gel and commercial cream formulations of CP.

The effect of methyl palmitate on treatment of experimental asthma.

OBJECTIVE: To determine the effects of methyl palmitate on murine model of chronic asthma. METHODS: The experimental study was conducted in the animal laboratory of DokuzEylul University, Turkey, from October to December, 2012, and comprised BALB/c mice whowere divided into four equal groups: three experimental and one control group. All groups except the control group were sensitised and challenged with ovalbumin. Mice with experimentally-induced asthma in Group I received saline; Group II dexamethasone 1mg/kg; Group III methyl palmitate300mg/kg intraperitoneally three times per week in the last four weeks of the study period. Animals were sacrificed 24h after the last administration of study drugs. Histological findings of airways were evaluated by light microscopic examination. Blood samples from vena cava inferior were taken for measurement of interleukin-5 levels. SPSS 15 was used for statistical analysis. RESULTS: The 28 female mice in the study were divided into 4 groups of 7(25%) each. The age range of the animals was 6-8weeks, and the weight range was 18-20g. All histological parameters and interleukin-5 levels of asthma in the Group III were significantly ameliorated compared to the Group I (p<0.05). All histological parameters and interleukin-5 levels were similar between Group III and Group II. CONCLUSIONS: Methyl palmitate exhibited anti-inflammatory effects by resolving the histological changes and reducing the interleukin-5 levels in murine model of chronic asthma.

Effects of lipoic acid in an experimentally induced hypertensive and diabetic rat model.

In this study, experimental diabetes and nephrectomy have been applied separately and together in order to investigate the possible therapeutic effects of lipoic acid (LA) on hypertensive and diabetic rat kidneys. Wistar rats were divided into eight groups: control, diabetes mellitus (DM), 5/6 nephrectomy, DM + 5/6 nephrectomy, LA administration, DM + LA treated, 5/6 nephrectomy + LA treated, and DM + 5/6 nephrectomy + LA-treated groups, respectively. Renal damage was evaluated histomorphometrically, ultrastructurally, and biochemically. Our findings supported that diabetes and hypertension together increased the rate of renal injury, and LA had therapeutic effects on hypertensive and diabetic rat kidneys.

The effect of insulin treatment on Rac1 expression in diabetic kidney.

This study was designed to evaluate the renoprotective effect of insulin on diabetic nephropathy through Rac1 inhibition. Twenty Wistar rats were divided into three groups: control (C), diabetic (D), and insulin-treated diabetic (D + I). Diabetes was induced by a single streptozotocin (STZ) injection (45 mg/kg i.p.) in adult male rats. Diabetic animals were treated subcutaneously with insulin (6 U/kg), or saline once a day for 8 weeks. Age-matched control rats received only saline. The kidney tissue samples were analyzed by immunohistochemical staining for Rac1 and cleaved caspase-3 expressions and using the TUNEL method for determining apoptotic cells. Diabetes increased the number of TUNEL (+) cells and cleaved caspase-3 and Rac1 expression levels in kidney. Administration of insulin for 8 weeks reduced Rac1 expression and ameliorated histopathological changes in kidney of STZ-induced diabetes model. These results may suggest that the renoprotective effect of insulin at least partly results from inhibition of Rac1 overexpression.

The effects of α-lipoic acid against testicular ischemia-reperfusion injury in Rats.

Testicular torsion is one of the urologic emergencies occurring frequently in neonatal and adolescent period. Testis is sensitive to ischemia-reperfusion injury, and, therefore, ischemia and consecutive reperfusion cause an enhanced formation of reactive oxygen species that result in testicular cell damage and apoptosis. α-lipoic acid is a free radical scavenger and a biological antioxidant. It is widely used in the prevention of oxidative stress and cellular damage. We aimed to investigate the protective effect of α-lipoic acid on testicular damage in rats subjected to testicular ischemia-reperfusion injury. 35 rats were randomly divided into 5 groups: control, sham operated, ischemia, ischemia-reperfusion, and ischemia-reperfusion +lipoic acid groups, 2 h torsion and 2 h detorsion of the testis were performed. Testicular cell damage was examined by H-E staining. TUNEL and active caspase-3 immunostaining were used to detect germ cell apoptosis. GPx , SOD activity, and MDA levels were evaluated. Histological evaluation showed that α-lipoic acid pretreatment reduced testicular cell damage and decreased TUNEL and caspase-3-positive cells. Additionally, α-lipoic acid administration decreased the GPx and SOD activity and increased the MDA levels. The present results suggest that LA is a potentially beneficial agent in protecting testicular I/R in rats.

Neuroprotective effects of resveratrol against traumatic brain injury in immature rats.

Childhood trauma resulting in traumatic brain injury (TBI) due to accidents and abuse is the major cause of death and dysfunction in the young. Since there are no approved specific pharmacological agents that block the progression of the secondary injury, the current management of TBI is mainly supportive. We aimed to determine the effect of resveratrol on hippocampal damage and behavioral deficits in 7-day-old rat pups subjected to contusion injury. Resveratrol was injected intraperitoneally at the doses of 100 mg/kg of body weight immediately after induction of traumatic injury. Hippocampal damage was examined by cresyl violet staining and behavioral alterations were evaluated using open field and novel object recognition tests 2 weeks after trauma. Histopathological evaluation showed that treatment with a single dose of 100 mg/kg resveratrol (i.p.) after the trauma significantly ameliorated the trauma induced hippocampal neuron loss at ipsilateral and contralateral hippocampal brain regions of rats. Additionally, treatment with resveratrol decreased anxiety and increased cortex/hippocampus dependent memory of animals subjected to blunt head trauma. These results show that acute treatment of resveratrol has a neuroprotective role against trauma induced hippocampal neuron loss and associated cognitive impairment in rats.

A new device to produce a standardized experimental fracture in the rat tibia.

OBJECTIVE: To develop and introduce a new device to produce a standardized closed experimental fracture in the rat tibia. METHODS: This study took place in the Research Laboratory, Medical Faculty, Dokuz Eylul University, in the year 2003. We include 20 healthy male white Wistar rats. After pinning both tibia of the rat intramedullary with the needle of a sterile injector without any incision, we tried to produce a fracture with the pendulum of the device, which was dropped in different angles in 9 rats. The tibial diaphysis of 14 rats in the main study were fractured at 60 degrees. After the fractures were confirmed radiologically, 4 tibia underwent pathological analysis to determine the degree of soft tissue damage and 24 tibia were examined in terms of histological fracture healing. RESULTS: Radiologically, this technique resulted in a transverse or short oblique bicortical fracture in the middle of the tibial diaphysis. The healing process was well adjusted with the classification of Allen. No noticeable soft tissue damage in the fracture region was demonstrated. CONCLUSION: This method of producing an easy and reproducible fracture in a standard fashion without displacement and minimal soft tissue trauma in laboratory animals with this simple apparatus make it a useful technique for bone healing studies.

Effect of dexmedetomidine on acute lung injury in experimental ischemia-reperfusion model.

PURPOSE: Ischemia-reperfusion injury is one of the consequences of tourniquet application for extremity surgery. The aim of the study was to establish the effect of dexmedetomidine on the acute lung injury following lower extremity experimental ischemia-reperfusion model in rats. METHODS: Twenty-eight Wistar-Albino breed Rats were recruited after Ethics Committee approval and allocated into 4 groups, each with 7 subjects. Group 1 (SHAM) received only anesthesia. Group 2 (IR) had experienced 3h of ischemia and 3h of reperfusion using left lower extremity tourniquet after anesthesia application. Groups 3 (D-50) and 4 (D-100) had undergone the same procedures as in the Group 2, except for receiving 50 and 100mg·kg-1, respectively, dexmedetomidine intraperitoneally 1h before the tourniquet release. Blood samples were obtained for the analysis of tumor necrosing factor-α and interleukin-6. Pulmonary tissue samples were obtained for histological analysis. RESULTS: No significant difference regarding blood tumor necrosing factor-α and interleukin-6 values was found among the groups, whereas pulmonary tissue injury scores revealed significant difference. Histological scores obtained from the Group 2 were significantly higher from those in the Groups 1, 3 and 4 with p-values 0.001 for each comparison. Moreover, Group 1 scores were found to be significantly lower than those in the Groups 3 and 4 with p-values 0.001 and 0.011, respectively. No significant difference was observed between the Groups 3 and 4. CONCLUSION: Dexmedetomidine is effective in reduction of the experimental ischemia-reperfusion induced pulmonary tissue injury in rats, formed by extremity tourniquet application.

Wound healing effects of collagen-laminin dermal matrix impregnated with resveratrol loaded hyaluronic acid-DPPC microparticles in diabetic rats.

An alternative formulation for the treatment of diabetic foot wounds that heal slowly is a requirement in pharmaceutical field. The aim of this study was to develop a dermal matrix consisting of skin proteins and lipids with an antioxidant that will enhance healing and balance the oxidative stress in the diabetic wound area due to the high levels of glucose. Thus a novel three dimensional collagen-laminin porous dermal matrix was developed by lyophilization. Resveratrol-loaded hyaluronic acid and dipalmitoylphosphatidylcholine microparticles were combined with this dermal matrix. Characterization, in vitro release, microbiological and in vivo studies were performed. Spherical microparticles were obtained with a high RSV encapsulation efficacy. The microparticles were well dispersed in the dermal matrix from the surface to deeper layers. Collagenase degraded dermal matrix, however the addition of RSV loaded microparticles delayed the degradation time. The release of RSV was sustained and reached 70% after 6h. Histological changes and antioxidant parameters in different treatment groups were investigated in full-thickness excision diabetic rat model. Collagen fibers were intense and improved by the presence of formulation without any signs of inflammation. The highest healing score was obtained with the dermal matrix impregnated with RSV-microparticles with an increased antioxidant activity. Collagen-laminin dermal matrix with RSV microparticles was synergistically effective due to presence of skin components in the formulation and controlled release achieved. This combination is a safe and promising option for the treatment of diabetic wounds requiring long recovery.

[Effect of dexmedetomidine on acute lung injury in experimental ischemia-reperfusion model]. / Efeito de dexmedetomidina sobre lesão pulmonar aguda em modelo experimental de isquemia­reperfusão.

PURPOSE: Ischemia-reperfusion injury is one of the consequences of tourniquet application for extremity surgery. The aim of the study was to establish the effect of dexmedetomidine on the acute lung injury following lower extremity experimental ischemia-reperfusion model in rats. METHODS: Twenty-eight Wistar-Albino breed rats were recruited after Ethics Committee approval and allocated into 4 groups, each with 7 subjects. Group 1 (SHAM) received only anesthesia. Group 2 (IR) had experienced 3h of ischemia and 3h of reperfusion using left lower extremity tourniquet after anesthesia application. Groups 3 (D-50) and 4 (D-100) had undergone the same procedures as in the Group 2, except for receiving 50 and 100mg.kg-1, respectively, dexmedetomidine intraperitoneally 1h before the tourniquet release. Blood samples were obtained for the analysis of tumor necrosing factor-α and interleukin-6. Pulmonary tissue samples were obtained for histological analysis. RESULTS: No significant difference regarding blood tumor necrosing factor-α and interleukin-6 values was found among the groups, whereas pulmonary tissue injury scores revealed significant difference. Histological scores obtained from the Group 2 were significantly higher from those in the Groups 1, 3 and 4 with p-values 0.001 for each comparison. Moreover, Group 1 scores were found to be significantly lower than those in the Groups 3 and 4 with p-values 0.001 and 0.011, respectively. No significant difference was observed between the Groups 3 and 4. CONCLUSION: Dexmedetomidine is effective in reduction of the experimental ischemia-reperfusion induced pulmonary tissue injury in rats, formed by extremity tourniquet application.

Effective topical delivery systems for corticosteroids: dermatological and histological evaluations.

Atopic dermatitis (AD) is a chronic and relapsing skin disease with severe eczematous lesions. Long-term topical corticosteroid treatment can induce skin atrophy, hypopigmentation and transepidermal water loss (TEWL) increase. A new treatment approach was needed to reduce the risk by dermal targeting. For this purpose, Betamethasone valerate (BMV)/Diflucortolone valerate (DFV)-loaded liposomes (220-350 nm) were prepared and incorporated into chitosan gel to obtain adequate viscosity (∼13 000 cps). Drugs were localized in stratum corneum + epidermis of rat skin in ex-vivo permeation studies. The toxicity was assessed on human fibroblast cells. In point of in-vivo studies, pharmacodynamic responses, treatment efficacy and skin irritation were evaluated and compared with previously prepared nanoparticles. Liposome/nanoparticle in gel formulations produced higher paw edema inhibition in rats with respect to the commercial cream. Similar skin blanching effect with commercial creams was obtained via liposome in gels although they contain 10 times less drug. Dermatological scoring results, prognostic histological parameters and suppression of mast cell numbers showed higher treatment efficiency of liposome/nanoparticle in gel formulations in AD-induced rats. TEWL and erythema measurements confirmed these results. Overview of obtained results showed that liposomes might be an effective and safe carrier for corticosteroids in skin disease treatment.

Expression and activity levels of matrix metalloproteinase-7 and in situ localization of caseinolytic activity in colorectal cancer.

OBJECTIVES: Matrix metalloproteinase-7 is capable of degrading several ECM and non-ECM molecules and contributes to colorectal cancer progression and metastasis. Here, we examined the significance of MMP-7 in colorectal tumors by detecting active and latent MMP-7 levels and localization of its caseinolytic activity. DESIGN AND METHODS: We investigated expression levels, localization, and proteolytic activity of MMP-7 and local caseinolytic activity in colorectal tumor and paired normal tissues by using real time PCR, casein zymography, immunohistochemistry and in situ casein zymography, respectively. In addition the results were compared with clinicopathological variables. RESULTS: Real time PCR and immunohistochemistry showed that MMP-7 expressions were higher in colorectal tumor tissues than in normal tissues. Also, mRNA expressions of MMP-7 were positively correlated with tumor and pathological stages and negatively correlated with age. Furthermore, MMP-7 mRNA expression had a sensitivity of 81.3% and a specificity of 81.2% at a cut-off value of 0.0006, making it a potential marker for diagnosis of colorectal cancer. According to casein zymography, pro- and active MMP-7 levels were also elevated in tumor tissues. In addition, we assessed local caseinolytic activity using in situ casein zymography. Increased immunoreactivity of MMP-7 and local caseinolytic activity were found in neoplastic cells but not in stromal cells. CONCLUSION: We emphasized the significant role of MMP-7 in diagnosis and progression and/or development of colorectal cancer.

Unfolded protein response prevents blastocyst formation during preimplantation embryo development in vitro.

OBJECTIVE: To study the effect of increased endoplasmic reticulum (ER) stress as a major nongenomic mechanism for arrested blastocyst development. DESIGN: Cell and animal study. SETTING: The Ohio State University and Yale University. ANIMAL(S): Mice. INTERVENTION(S): Pregnant mare serum gonadotropin and hCG were administered IP; two cell embryos were collected 48 hours after hCG administration. MAIN OUTCOME MEASURE(S): Blastocyst development rate. RESULT(S): No morphological difference was detected in control versus tunicamycin- (TM) treated embryos until the blastocyst stage. On day 4 of embryonic development, TM treatment reduced blastocyst formation from 79% to 4% and induced nuclear fragmentation. TM treatment caused 2-fold and 2.6-fold increase in binding immunoglobulin protein and spliced-X-box binding protein 1 mRNA expression, respectively. By comparison, the tauroursodeoxycholic acid + TM combination reversed the effect of TM alone on blastocyst formation to near control levels. CONCLUSION(S): These results indicate that increased ER stress during in vitro embryo development triggers an unfolded protein response (UPR) that negatively affects blastocyst formation and suggests that activation of UPR signaling may account for low rates of blastocyst development.

Protective Effects of Resveratrol against Chronic Immobilization Stress on Testis.

Objective. The aim of this study was to investigate protective effects of resveratrol, a strong antioxidant, against possible negative effects of chronic immobilization stress on testes of male rats histochemically, immunohistochemically, ultrastructurally, and biochemically. Material and Methods. Male Wistar rats were divided into 4 groups (n = 7). Group I, control group (C), was not exposed to stress. Group II, stress group (S), was exposed to chronic immobilization stress. In Group III, low dose resveratrol + stress group (LRS), rats were given 10 mg/kg/day resveratrol just before the stress application. In Group IV, high dose resveratrol + stress group (HRS), rats were given 20 mg/kg/day resveratrol just before the stress application. For chronic immobilization stress application animals were put in the plastic tubes (6 cm in diameter, 15 cm in length) during 32 days for 6 hours. All animals were sacrificed 18 hours after the last stress application. Results. Histochemical and ultrastructural investigations showed that in stress group there was germ cell deprivation in seminiferous tubules and increase of connective tissue on interstitial area. No significant changes were seen in low and high dose resveratrol groups. After immunohistochemical investigations, TUNEL (+) and Active Caspase-3 (+) cells were increased in seminiferous tubules of stress group compared with those control group, but they were decreased in low and high dose resveratrol groups. According to biochemically results, MDA, GSH, and testosterone levels in stress group showed no significant difference when compared with those of the other groups. Conclusion. The chronic immobilization stress increases oxidative stress and apoptosis and causes histological tissue damages; resveratrol can minimize the histological damage in testes significantly.

Deoxycholate hydrogels of betamethasone-17-valerate intended for topical use: In vitro and in vivo evaluation.

The aim of this study was to evaluate the suitability of sodium-deoxycholate (Na-DOC) gels containing betamethasone-17-valerate (BMV) for topical application. The gels were characterized for rheological and textural properties. The in vitro flux of BMV from Na-DOC gels across rat skin was 2.5 (0.05% gel) and 8.5 times (0.1% gel) higher compared to the commercial cream (0.1%), respectively. The pharmacodynamic responses after in vivo topical application in rats were also determined. A significant correlation between anti-inflammatory activity and in vitro permeation of BMV was observed. Na-DOC gels produced significantly higher edema inhibition compared to commercial cream at all time intervals. Finally, according to the results of histology studies, Na-DOC gel has no irritant effect on the skin. In conclusion, Na-DOC gel formulation could be suggested as a promising alternative system for the topical application of BMV.

The protective effects of carnosine and melatonin in ischemia-reperfusion injury in the rat liver.

The reperfusion following liver ischemia results in hepatocyte damage and apoptosis. The aim of this study was to investigate the effects of two antioxidant agents, carnosine and melatonin, in rat liver ischemia-reperfusion injury. Five study groups were formed; I. sham, II. ischemia-reperfusion, III. ischemia-reperfusion+melatonin, IV. ischemia-reperfusion+carnosine, V. ischemia-reperfusion+melatonin+carnosine. Then 250 mg/kg carnosine and 10 mg/kg melatonin were administered intraperitoneally 30 min before ischemia and immediately after the reperfusion. Sinusoidal dilatation, congestion and neutrophil infiltration were observed in the ischemia-reperfusion group while these symptoms were less pronounced in the treatment groups. Alanine aminotransferase, aspartate aminotransferase and myeloperoxidase levels were increased in the ischemia-reperfusion group while they were lowered in the treatment groups. Glutathione level was low in the ischemia-reperfusion group while it tended to increase in the ischemia-reperfusion+carnosine administered and ischemia-reperfusion+carnosine+melatonin administered groups. There was an increase in the number of apoptotic cells in the ischemia-reperfusion group while this number was lowered in the treatment groups. Carnosine was more effective than melatonin in the reversal of structural and biochemical alterations that resulted from ischemia-reperfusion injury. The administration of melatonin and carnosine together yielded better outcomes compared to the sole administration of each agent.

The effects of electromagnetic fields on the ultrastructure of heart / Efectos de los campos electromagnéticos en la ultraestructura del corazón

Increasingly the use and convenience of electrical appliances in our daily lives are the cause of harmful effects caused by electromagnetic fields (EMF). The aim of this study was to research the effect of EMF on the ultrastructure of the heart in EMF exposed rats. In this study 45 male Sprague Dawley rats ranging in weight between 260 and 280 grams were used. The rats were divided into 3 groups, control (n:15), Sham (n:15) and EMF group (n: 15) and exposed for 14 days 3 hours per day; gauss levels at 2.5 were applied to the EMF group, while the sham group in the same environment in Plexiglas cage was kept for 14 days 3 hours per day without magnetic field exposure. Control group at 14/10 hours light dark cycle fed in normal cages for 14 days. After two weeks rats were sacrificed by 50mg/kg of Ketalar anesthesia and heart tissue fixed in 2.5 gluteraldehide. Routine follow up with electron microscopic assessment. Mitochondrial structures and cellular structures observed in all the groups were normal. Myofibrillar loss, dilatation of smooth endoplasmic reticulum, mitochondrial swelling or cristalysis was not observed. Intercalated disc degeneration and apoptosis of nucleus was not observed. Therefore, and as a result of our study we did not observe differences between control and EMF groups.

El uso y la comodidad de los aparatos eléctricos en nuestra vida cotidiana cada vez más son causa de efectos perjudiciales debido a los campos electromagnéticos(CEM).El objetivo de este estudio fue investigar el efecto de los CEM sobre la ultraestructura del corazón en ratas. Fueron utilizadas 45ratas Sprague Daw ley, con peso entre 260 y 280gramos. Las ratas fueron divididas en 3 grupos: control (n: 15); Sham (n:15), y grupo expuesto a CEM (n:15) durante 14 días,3 horas por día. Se aplicó niveles de 2,5gaussal grupo expuesto a CEM, mientras que el grupo de tratamiento simulado en el mismo entorno en jaulas plexiglás se mantuvo durante 14 días 3 horas día, sin exposición a campo electromagnético. Grupo control alimentado en jaulas normales durante 14 días con ciclo luz/oscuridad de 14/10. Al termino de dos semanas las ratas fueron sacrificadas por medio de anestesia Ketalar 50mg/kg y el tejido del corazón fijado engluteraldehido al 2,5. Se realizó seguimiento de rutina con correspondiente evaluación de microscopía electrónica. Las estructuras mitocondriales y celular es observadas en todos los grupos eran normales. No se observó pérdida miofibrilar, tampoco aumento del volumen mitocondrial ni dilatación del retículo endoplásmico lisoocristalysis. No se observó degeneración de los discosintercaladoso apoptosis de núcleo. Por lo tanto,y como resultado de nuestro estudio no encontramos diferencias entre los grupos control y CEM.