Azoxymethane-induced carcinogenesis-like model of mouse intestine and mouse embryonic stem cell-derived intestinal organoids.

BACKGROUND: Tumor modeling using organoids holds potential in studies of cancer development, enlightening both the intracellular and extracellular molecular mechanisms behind different cancer types, biobanking, and drug screening. Intestinal organoids can be generated in vitro using a unique type of adult stem cells which are found at the base of crypts and are characterized by their high Lgr5 expression levels. METHODS AND RESULTS: In this study, we successfully established intestinal cancer organoid models by using both the BALB/c derived and mouse embryonic stem cells (mESCs)-derived intestinal organoids. In both cases, carcinogenesis-like model was developed by using azoxymethane (AOM) treatment. Carcinogenesis-like model was verified by H&E staining, immunostaining, relative mRNA expression analysis, and LC/MS analysis. The morphologic analysis demonstrated that the number of generated organoids, the number of crypts, and the intensity of the organoids were significantly augmented in AOM-treated intestinal organoids compared to non-AOM-treated ones. Relative mRNA expression data revealed that there was a significant increase in both Wnt signaling pathway-related genes and pluripotency transcription factors in the AOM-induced intestinal organoids. CONCLUSION: We successfully developed simple carcinogenesis-like models using mESC-based and Lgr5 + stem cell-based intestinal organoids. Intestinal organoid based carcinogenesi models might be used for personalized cancer therapy in the future.

Experimental Study: The Effect Of 20% Intralipid Emulsion Therapy In Cerebral Ischemic Reperfusion.

BACKGROUND: This experimental study was conducted to investigate the effect of 20% Intralipid Emulsion (ILE) treatment on Cerebral Ischemia Reperfusion Injury (CIRI) after reperfusion in acute ischemic stroke. METHODS: In this experimental study, seven rats without any intervention (control group), seven rats (sham group) for which CIRI was created after the common carotid artery was ligated for 2 hours, and seven rats who were treated with 20% ILE after CIRI (CIRI + ILE group) were sacrificed after 24 hours, and histopathological findings were investigated. RESULTS: In rats that were not treated after CIRI, 52.7% had level-1, 32.7% had level-2. and 14.5% had level-3. histopathological findings. While 72.2% of the rats treated with ILE had level-1 and 27.8% had level-2 findings, no level-3 histopathological findings were detected in any of the rats. While no signs of coagulative necrosis, spongiosis of surrounding tissue and polymorphonuclear leukocytes were observed histopathological in any of the rats given ILE, there was no macrophages finding in 85.6% of the rats. ILE treatment also reduced the histopathological findings of eosinophilic neurons, astrogliosis, neovascularization, vascular thrombosis and mononuclear inflammatory cells. CONCLUSION: This study showed that 20% ILE treatment reduces the histopathological damage seen in cerebral ischemia and CIRI.

Safety assessment of European cranberrybush (Viburnum opulus L.) fruit juice: Acute and subacute oral toxicity.

European cranberrybush (ECB) (Viburnum opulus L.) fruits are abundant in phenolic compounds associated with various health benefits. However, the toxicity and safety of ECB juice have not been systematically studied. In the present study, acute and subacute oral toxicities of ECB fruit juice were evaluated on Sprague-Dawley rats and BALB/c mice to establish a toxicity profile. In acute tests, a single administration of 2000 mg/kg body weight of extract to rats exhibited no clinical signs of toxicity or mortality, indicating that the lethal dose (LD50) was over 2000 mg/kg. In subacute tests, repeated administration for 28 days at 0 (control), 500, and 2000 mg/kg doses of extract in mice did not display adverse clinical signs or deaths. However, in the 2000 mg/kg subacute group, platelet counts were significantly high, which correlated with histopathological analyses revealing that ECB extract at 2000 mg/kg was toxic to the kidney, liver, and adipose tissue. The NOAEL value of ECB extract was found as 500 mg/kg/day, but further sub-chronic and chronic toxicity studies are warranted to comprehensively evaluate the long-term safety implications. The study's results emphasize the importance of considering the dosage of dietary supplements containing high levels of phenolic compounds over an extended period to avoid potential cumulative effects from prolonged consumption of high doses.

Nanoemulsions as a Promising Carrier for Topical Delivery of Etodolac: Formulation Development and Characterization.

This research primarily focuses on the development of innovative topical nanoemulsions for etodolac, aimed at surmounting its inherent limitations. The preparation of etodolac nanoemulsions is accomplished through a combination of high shear homogenization and ultrasonication methods. The optimization of the formulation components is systematically conducted using the design of experiments methodology. The droplet size (DS), polydispersity index (PDI), and zeta potential (ZP) of the optimized formulation were assessed using the differential light scattering (DLS) technique. Surface morphology examinations were conducted using electron microscopy, while interactions between excipients and the drug were analyzed through FTIR analysis. Additionally, in vitro release and ex vivo permeability studies were carried out. Furthermore, anti-inflammatory activity was evaluated in the context of a carrageenan-induced paw edema model in rats. The DS, PDI, and ZP of the optimal formulation were 163.5 nm, 0.141, and -33.1 mV, respectively. The in vitro release profile was assessed as a sustained release by following a non-Fickian drug transport. The flux of etodolac nanoemulsions and coarse dispersions were 165.7 ± 11.7 µg/cm2 h and 59.7 ± 15.2 µg/cm2 h, respectively. Enhanced edema inhibition was observed at 13.4%, 36.5%, and 50.65% for the 6th, 8th, and 24th hours, respectively. Taken together, these results confirmed that nanoemulsions are promising carriers for the topical delivery of etodolac.

Effect of methylphenidate on the onset of puberty and reproductive organ development in rats.

Methylphenidate (MPH) is the first-line therapy for attention deficit hyperactivity disorder (ADHD) in children and adolescents. The aim of this study was to investigate the effects chronic MPH administration on reproductive parameters in both male and female pre-pubertal rats and reversibility of these effects. Sprague-Dawley rats were administered with 5 mg/kg MPH or saline orally from postnatal day (PND) 21 to PND60 and from PND21 to PND90. In addition, recovery groups from both sexes, in which MPH administration was stopped from PND60 to PND90 were included. Puberty onset, serum luteinizing hormone (LH), follicle stimulating hormone (FSH), testosterone and estradiol levels were determined. Histopathology of male and female reproductive organs was examined. Puberty onset was significantly early in the males (p<0.01), but late in females (p<0.05). In males, serum LH and FSH levels were similar. Testosterone levels tended to decrease in MPH-treated animals. Morphology of testes, epididymis and vas deferens was disrupted in MPH-treated animals, while it was improved in the recovery group. In females, estradiol levels decreased in MPH-treated group compared to controls, and elevated LH levels were detected in recovery group. Similar to the males, disruption in the reproductive organ histology was seen with morphological deterioration in basement membrane of the ovaries of MPH-treated groups. These adverse effects of MPH were recovered after drug cessation for 30 days. The present results demonstrate that MPH could affect the reproductive functions in both male and female rats. However, our findings also suggest that those effects are transient.

Design and characterization of dexamethasone loaded microsponges for the management of ulcerative colitis.

Ulcerative colitis is an inflammatory condition with ulcerations throughout the colon. The existing remedies have some limitations such as drug inactivation, poor absorption, and adverse reactions. The present study aimed to design novel microsponge formulations to enhance remission of the dexamethasone (as a model pharmaceutical ingredient) in the colon. Microsponges were prepared by using the quasi-emulsion technique. The optimal formulation was selected by applying the design of experiments approach which used methylcellulose (MC) (0.75-2%, w/w), polyvinylalcohol (PVA)(0.5-1%, w/w), and tween 80 (TW80) (1.5-2.5%, w/w). The critical quality attributes were selected as particle size and entrapment efficiency. The particle size and encapsulation efficiency were found as 140.38 ± 9.2 µm and 77.96 ± 3.4 %. After the optimization; morphological, thermal, and physicochemical characterization studies were performed. Ultimately, the optimal formulation was investigated by using the acetic acid-induced ulcerative colitis model in rats. The physicochemical characterization studies confirmed that the formulation components were compatible with each other. The in vitro release mechanisms were fitted to First order kinetics at pH 1.2 (R2:0.9563), and Korsmeyer-Peppas kinetics at pH 4.5 (R2: 0.9877), and pH 6.8 (R2: 0.9706). The medicated microsponges exhibited remarkable recovery compared to the control group of the in vivo ulcerative colitis model (p < 0.05). It could be concluded that microsponges were evaluated as a promising alternative drug delivery system for the management of ulcerative colitis.

Determination of In Vivo efficacy and safety of zeolite as a new pleurodesis agent.

Pleural effusion, the pathological condition in which an abnormal amount of pleural fluid is accumulated in the small space between the visceral and parietal pleurae of the lungs, can be treated by pleurodesis, whereby the pleural space is obliterated. This effect can be achieved by chemical pleurodesis utilizing various reagents such as talc, an agent commonly employed in pleurodesis. Zeolites, microporous tectosilicates found in nature as minerals, can be used in a wide range of medical applications. Different zeolite compounds may exhibit variable efficacy and safety profiles, mainly depending on their particle size. In this study, we evaluated the efficacy and safety of zeolite pleurodesis. New Zealand rabbits were administered 400 mg/kg of either agent dissolved in 2 mL of isotonic saline solution by injection into their pleural cavity, and computed tomography images were obtained on postoperative day 26. Euthanization was conducted at the end of 28 days for histopathological evaluation. Furthermore, subacute toxicity and mutagenicity profiles of zeolite were analyzed. Our findings revealed that zeolite was able to induce an adequate inflammatory response to achieve successful pleurodesis. The adhesion profiles were in favor of zeolite when compared to talc pleurodesis. Moreover, none of the tested doses of zeolite induced subacute toxicity or mutagenesis. Collectively, our results suggested zeolite as an effective and safe pleurodesis agent.

What is the protective effect of preischemic kisspeptin-10 administration against ischemia/reperfusion injury of striatum on mice?

BACKGROUND: Kisspeptin is a neuropeptide with a primary role on the onset of puberty and has beneficial effects on ischemia/ reperfusion (I/R) injury. In this study, we aimed to investigate the effect of kisspeptin administration on striatal I/R injury in mice. METHODS: Forty adult C57/BL6 mice were randomly divided into four groups: Sham, Kisspeptin, I/R, and I/R + Kisspeptin groups. The groups were administered with either physiological saline (Sham and I/R groups) or kisspeptin (Kisspeptin and I/R + Kisspeptin groups) intraperitoneally 40 min before the operation. A microdialysis probe was placed in the right striatum according to stereotaxic coordinates. During the experimental period, artificial cerebrospinal fluid was passed through the micropump. Then, transient cerebral ischemia was established by compressing both common carotid arteries with an aneurysm clip for 15 min and animals were reperfused for 2 h. Throughout the process of microdialysis (before, during and after I/R period), samples were collected to measure dopamine (DA), noradrenaline (NA), and 3,4-dihydroxyphenylglycine (DHPG) at intervals of 20 min continuously. At the end of the reperfusion period, the animals were decapitated, striatum was dissected, half of the animals were used for oxidative stress analyses (reduced glutathione, glutathione S-transferase (GST), superoxide dismutase (SOD), malondialdehyde (MDA), and the other half were used for histopathology analyses. RESULTS: Number of glial cells was significantly increased in kisspeptin-administered groups. DA levels in ischemic animals were decreased by kisspeptin administration (p < 0.0001). NA levels were reduced in animals administered with kisspeptin without I/R injury (p < 0.05). DHPG levels reduced during the reperfusion period in ischemic animals (p < 0.05). Kisspeptin did not exhibit a significant antioxidant activity in the ischemic animals, while GST and SOD levels were reduced in the I/R + kisspeptin group compared to the kisspeptin group (p < 0.05). DISCUSSION: Our results suggest that kisspeptin may be regulating the neurotransmitter release and metabolism, as well as inflammatory response in brain upon I/R injury.

Investigation of obesogenic effects of hexachlorobenzene, DDT and DDE in male rats.

Obesity has become a very important public health problem and is increasing globally. Genetics, individual and environmental factors play roles in the etiology of this complex disorder. Recently, several environmental pollutants have been suggested to have obesogenic activities. Peroxisome proliferator activating receptor gamma (PPARγ), uncoupling protein-1 (UCP1) and their expression in white adipose tissue (WAT) and brown adipose tissue (BAT) play key roles in adipogenesis. UCP3 and irisin were reported to play roles in non-shivering thermogenesis. Our primary aim was to investigate obesogenic effects of hexachlorobenzene (HCB), dichlorodiphenyltrichloroethane (DDT) and dichlorodiphenyldichloroethylene (DDE) in rats. In addition, thermoregulatory effects of HCB, DDT and DDE were also investigated by analyzing the levels of Ucp3 and irisin. Thirty-two adult male Sprague-Dawley rats were randomly divided into four groups as control, HCB, DDT and DDE. Animals were administered with organochlorine pesticides (OCPs; 5 mg/kg bw) by oral gavage every other day for five weeks. At the end of the experimental period, the animals were sacrificed, BAT and WAT samples were collected to analyze Pparγ, Ucp1 and Ucp3 levels. Moreover, skeletal muscle samples were collected to examine Ucp3 and irisin levels. Serum glucose, cholesterol and triglyceride levels were also determined. Body weight and core temperature of the animals were not significantly affected by any of the OCP administration. Serum glucose, cholesterol and triglyceride levels were similar among the experimental groups. Pparγ expression was significantly elevated by HCB administration only in WAT (p < 0.05). On the other hand, both Pparγ and Ucp1 expressions were diminished in WAT and BAT (p < 0.01) by DDT treatment, while in WAT, DDE significantly decreased Pparγ expression without altering its expression in BAT (p < 0.001). Ucp3 and irisin levels in skeletal muscle were not altered. Our findings show that both DDT and DDE reduce the browning of WAT by suppressing white adipocytes and thus may have obesogenic activity in male rats without altering thermoregulation. In addition, HCB, DDT and DDE-induced alterations in expression of Pparγ and Ucp1 in WAT implicates differential regulation of adipogenic processes.

Human ESC-derived Neuromesodermal Progenitors (NMPs) Successfully Differentiate into Mesenchymal Stem Cells (MSCs).

Mesenchymal Stem Cells (MSCs), as an adult stem cell type, are used to treat various disorders in clinics. However, derivation of homogenous and adequate amount of MSCs limits the regenerative treatment potential. Although mesoderm is the main source of mesenchymal progenitors during embryonic development, neuromesodermal progenitors (NMPs), reside in the primitive streak during development, is known to differentiate into paraxial mesoderm. In the current study, we generated NMPs from human embryonic stem cells (hESC), subsequently derived MSCs and characterized this cell population in vitro and in vivo. Using a bFGF and CHIR induced NMP formation protocol followed by serum containing culture conditions; here we show that MSCs can be generated from NMPs identified by not only the expression of T/Bra and Sox 2 but also FLK-1/PDGFRα in our study. NMP-derived MSCs were plastic adherent fibroblast like cells with colony forming capacity and trilineage (osteo-, chondro- and adipo-genic) differentiation potential. In the present study, we demonstrate that NMP-derived MSCs have an endothelial tendency which might be related to their FLK-1+/PDGFRα + NMP origin. NMP-derived MSCs displayed a protein expression profile of characterized MSCs. Growth factor and angiogenesis related pathway proteins were similarly expressed in NMP-derived MSCs and characterized MSCs. NMP-derived MSCs keep characteristics after short-term and long-term freeze-thaw cycles and localized into bone marrow followed by tail vein injection into NOD/SCID mice. Together, these data showed that hESC-derived NMPs might be used as a precursor cell population for MSC derivation and could be used for in vitro and in vivo research.

Comparative biochemical and histopathological evaluations proved that receptacle is the most effective part of Cynara scolymus against liver and kidney damages.

ETHNOPHARMACOLOGICAL RELEVANCE: The liver and kidney are among the most important organs in the body, where metabolic and elimination functions take place. During this process, liver and kidneys may suffer damage due to ingestion or formation of toxic metabolites leading to organ loss and even death. Artichoke (Cynara scolymus L.) leaf has long been recognized as a popular herbal remedy in traditional medicines with beneficial effects on liver. AIM OF THE STUDY: In phytotherapy leaves are the part used to support the liver functions and for treatment of damage induced by various toxins, while fleshy receptacle is cooked as meal to support liver homeostasis. However, effects of other plant parts on liver such as stems, bracts have not much attracted the attention of scientific community so far. In this study we investigated comparatively the hepatoprotective and nephroprotective effects of different plant parts of artichoke, i.e. receptacles, outer bracts, inner bracts, and stems with that of leaves upon paracetamol-induction in rats. MATERIALS AND METHODS: Aqueous ethanol (80%) extracts obtained from the different parts of artichoke were administered for five consecutive days after paracetamol induction to rats. At the end of experimental period blood samples from the experimental animals were taken for biochemical tests, while livers and kidneys were removed for further histopathological evaluation. RESULTS: The histopathological examinations of liver and kidney tissues revealed that the receptacle and stem extracts of the artichoke were the most effective parts by improving the experimentally induced pathology in both liver and kidney. Biochemical tests also supported the histopathological data; receptacle, stem and bract extracts reduced serum alanine transaminase (ALT) and aspartate transaminase (AST) levels, but not alkaline phosphatase (ALP), creatinine and blood urea nitrogen (BUN) levels. CONCLUSIONS: Histopathological and biochemical studies have shown that receptacle and stem extracts of artichoke were found to exert higher protective activity on liver and kidney damage induced by paracetamol comparing to its bract and leaf extracts, the latest is officially recognized as herbal remedy.

The Effects of Intravenous Lipid Emulsion Therapy in the Prevention of Depressive Effects of Propofol on Cardiovascular and Respiratory Systems: An Experimental Animal Study.

Background and objectives: Although there are several hypotheses about the mechanism of action, intravenous lipid emulsion (ILE) therapy has been shown to be effective in the treatment of toxicities due to local anaesthetics and many lipophilic drugs. In this study, we had hypothesized that ILE therapy might also be effective in preventing mortality and cardiorespiratory depressant effects due to propofol intoxication. Materials and methods: Twenty-eight Sprague-Dawley adult rats were randomly divided into four groups. Saline was administered to the subjects in the control group. The second group was administered propofol (PP group); the third group was administered ILE (ILE group), and the fourth group was administered propofol and ILE therapy together (ILE+PP group). Systolic blood pressure (SBP), diastolic blood pressure (DBP), respiratory rate (RR), heart rate (HR), and mortality were recorded at 10 time-points during a period of 60 min. A repeated measures linear mixed-effect model with unstructured covariance was used to compare the groups. Results: In the PP group; SBP, DBP, RR, and HR levels declined steadily; and all rats in this group died after the 60-min period. In the ILE+PP group, the initially reduced SBP, DBP, RR, and HR scores increased close to the levels observed in the control group. The SBP, DBP, RR, and HR values in the PP group were significantly lower compared to the other groups (p < 0.01). The mortality rate was 100% (with survival duration of 60 min) for the PP group; however, it was 0% for the remaining three groups. Conclusions: Our results suggest that the untoward effects of propofol including hypotension, bradycardia, and respiratory depression might be prevented with ILE therapy.

The Role of Intralipid Emulsion in the Rat Model of Digoxin Intoxication.

Although the mechanism of action is not well known, intravenous lipid emulsion (ILE) has been shown to be effective in the treatment of lipophilic drug intoxications. It is thought that, ILE probably separates the lipophilic drugs from target tissue by creating a lipid-rich compartment in the plasma. The second theory is that ILE provides energy to myocardium with high-dose free fatty acids activating the voltage-gated calcium channels in the myocytes. In this study, effects of ILE treatment on digoxin overdose were searched in an animal model in terms of cardiac side effects and survival. Forty Sprague-Dawley rats were divided into five groups. As the pre-treatment, the groups were administered saline, ILE, DigiFab and DigiFab and ILE. Following that, digoxin was infused to all groups until death except the control group. First arrhythmia and cardiac arrest observation times were recorded. According to the results, there was no statistically significant difference among the group in terms of first arrhythmia time and cardiac arrest times. However, when the saline group compared with ILE-treated group separately, significant difference was observed. DigiFab, ILE or ILE-DigiFab treatment make no significant difference in terms of the first arrhythmia and cardiac arrest duration in digoxin-intoxicated rats. However, it is not possible to say that at the given doses, ILE treatment might be successful at least as a known antidote. The fact that the statistical significance between the two groups is not observed in the subgroup analysis, the study should be repeated with larger groups.