Fonctionnalité in vivo d’un endothélium cornéen reconstitué par injection de cellules endothéliales cornéennes dans la chambre antérieure d’un modèle félin

Introduction : Malgré leur état non-prolifératif in vivo, les cellules endothéliales cornéennes (CEC) peuvent être amplifiées in vitro. Leur transplantation subséquente par injection intracamérale pourrait surmonter la pénurie de tissus associée à l’allo-greffe traditionnelle – l’unique traitement définitif disponible pour les endothéliopathies cornéennes. Objectif : Évaluer la fonctionnalité d’un endothélium cornéen reconstitué par injection de CEC dans la chambre antérieure du félin. Méthodes : Les yeux droits de 16 animaux ont été opérés. Huit ont été désendothélialisés centralement avec injection de 2x10e5 (n=4) ou 1x10e6 (n=4) CEC félines supplémentées avec Y-27632 et marquées avec SP-DiOC18(3). Deux ont été désendothélialisés complètement et injectés avec 1x10e6 CEC et Y-27632. Six contrôles ont été désendothélialisés centralement (n=3) ou complètement (n=3) et injectés avec Y-27632 sans CEC. La performance clinique, l’intégrité anatomique, le phénotype fonctionnel et l’expression de SP-DiOC18(3) du nouvel endothélium ont été étudiés. Résultats : Les cornées greffées avec 2x10e5 CEC et les contrôles désendothélialisés centralement ont réussi le mieux cliniquement. Les contrôles désendothélialisés complètement sont restés opaques. L’histopathologie a révélé une monocouche endothéliale fonctionnelle dans les cornées greffées avec 2x10e5 CEC et les contrôles désendothélialisés centralement, une multicouche endothéliale non-fonctionnelle dans les cornées désendothélialisées centralement et greffées avec 1x10e6 CEC, et un endothélium fibrotique non-fonctionnel dans les cornées désendothélialisées complètement. L’expression de SP-DiOC18(3) était rare dans les greffes. Conclusion : La thérapie par injection cellulaire a reconstitué un endothélium partiellement fonctionnel, auquel les CEC injectées n’ont contribué que peu. L’injection de Y-27632 sans CEC a reconstitué l’endothélium le plus sain. Des études additionnelles investiguant l’effet thérapeutique de Y-27632 seul sont justifiées.

Personalization of Electronic Nicotine Delivery Systems Toxicological impact in in-vivo model

Electronic nicotine delivery systems (ENDS) use has recently grown. E-cig generates carcinogenic chemical compounds and reactive oxygen species (ROS). Carbonyls and ROS are formed when the liquid comes into contact with the heating element. In this study the chemical and biological effects of coil resistance applied on the same device were investigated. A preliminary in-vivo study the new heat-not-burn devices (IQOS®) has been conducted to evaluate the effect of the device on antioxidant biomarkers. The amount of formaldehyde, acetaldehyde, acrolein was measured by GC-MS analysis. The two e-liquids used for carbonyls detection differed only for the presence of nicotine. The nicotine-free liquid was then used for the detection of ROS in the aerosol. The impact of the non-nicotine vapor on cell viability in H1299 human lung carcinoma cells, as well as the biological effects in a rat model of e-cig aerosol exposure, were also evaluated. After the exposure of Sprague Dawley rats to e-cig and IQOS® aerosol, the effect of 28-day treatment was examined on enzymatic and non-enzymatic antioxidant response, lung inflammation, blood homeostasis and tissue damage by using scanning electron microscope (SEM) technique. The results show a significant correlation between the low resistance and the generation of higher concentrations of the selected carbonyls and ROS in aerosols. Cell viability was reduced with an inverse relation to coil resistance. The experimental model highlighted an impairment of the pulmonary antioxidant and detoxifying machinery. Frames from SEM show disorganization of alveolar and bronchial epithelium. IQOS® exposed animals shows a significant production of ROS related to the unbalance of antioxidant defense and alteration of macromolecule integrity. This research demonstrates how several toxicological aspects can potentially occur in e-cig consumers who use low resistance device coupled with nicotine-free liquid. ENDS may expose users to hazardous compounds, which, may promote chronic pathologies and degenerative diseases.

Tecniche in vivo ancillari alla chirurgia di Mohs: ruolo delle nuove tecnologie nella valutazione dei margini chirurgici delle neoplasie cutanee maligne

Le neoplasie cutanee di tipo non-melanoma (non-melanoma skin cancers, NMSCs), quali il carcinoma a cellule basali (basal cell carcinoma, BCC) e il carcinoma a cellule squamose (squamous cell carcinoma, SCC) possono mostrare invasività locale e alto tasso di recidiva. La chirurgia microscopicamente controllata di Mohs (Mohs micrographic surgery, MMS) permette di eseguire una valutazione istologica immediata dei margini chirurgici delle neoplasie contestualmente alla loro escissione. Nel nostro studio abbiamo valutato del ruolo delle tecnologie in vivo (dermatoscopia e microscopia confocale a riflettanza, MCR) nella definizione dei margini preoperatori di NMSC ad alto rischio del volto e descritto la nostra esperienza con chirurgia tradizionale e MMS. Sono stati valutati 234 pazienti operati nel triennio 2019-2021: 39 con MMS e guida videodermatoscopica (Gruppo 1) e 195 con chirurgia tradizionale e guida videodermatoscopica (Gruppo 2). I pazienti operati nel periodo 2013-2018 (con MMS, Gruppo 3 (n = 241), e con chirurgia tradizionale, Gruppo 4 (n = 1086)) sono stati usati come confronto. La radicalità chirurgica è stata ottenuta nel Gruppo 1 nel 92,3% dei casi, con 1,2 steps in media di MMS (versus 1,7 nel Gruppo 3), nel Gruppo 2 nell’84,5% dei casi. La percentuale di non radicalità è stata: 7,7% nel Gruppo 1, 15,9% nel Gruppo 2, 6,2% nel Gruppo 3, 17,9% nel Gruppo 4. I tassi di recidiva sono stati: 5,1% nel Gruppo 1, 3,6% nel Gruppo 2, 4,1% nel Gruppo 3, 5,9% nel Gruppo 4, soprattutto in BCC di tipo sclerodermiforme e infiltrante. La MCR prechirurgica è stata utilizzata in 11 pazienti, con alcuni limiti nel delineare BCC di tipo sclerodermiforme e infiltrante. In conclusione, la videodermatoscopia e la MCR appaiono valide tecniche ancillari alla MMS e anche alla chirurgia tradizionale nel trattamento dei NMSCs. La MMS appare indicata soprattutto nei pazienti giovani e salvaguarda l’outcome estetico e funzionale.

MR in vivo tractography for the reconstruction of cranial nerves course

Aim The aim of my Ph.D. was to implement a diffusion tensor tractography (DTT) pipeline to reconstruct cranial nerve I (olfactory) to study COVID-19 patients, and anterior optic pathway (AOP, including optic nerve, chiasm, and optic tract) to study patients with sellar/parasellar tumors, and with Leber’s Hereditary Optic Neuropathy (LHON). Methods We recruited 23 patients with olfactory dysfunction after COVID-19 infection (mean age 37±14 years, 12 females); 27 patients with sellar/parasellar tumors displacing the optic chiasm eligible for endonasal endoscopic surgery (mean age 53. ±16.4 years, 13 female) and 6 LHON patients (mutation 11778/MT-ND4, mean age 24.9±15.7 years). Sex- and age-matched healthy control were also recruited. In LHON patients, optical coherence tomography (OCT) was performed. Acquisitions were performed on a clinical high field 3-T MRI scanner, using a multi-shell HARDI (High Angular Resolution Diffusion Imaging) sequence (b-values 0-300-1000-2000 s/mm2, 64 maximum gradient directions, 2mm3 isotropic voxel). DTT was performed with a multi-tissue spherical deconvolution approach and mean diffusivity (MD) DTT metrics were compared with healthy controls using an unpaired t-test. Correlations of DTT metrics with clinical data were sought by regression analysis. Results In all 23 hypo/anosmic patients with previous COVID-19 infection the CN I was successfully reconstructed with no DTT metrics alterations, thus suggesting the pathogenetic role of central olfactory cortical system dysfunction. In all 27 patients with sellar/parasellar tumors the AOP was reconstructed, and in 11/13 (84.7%) undergoing endonasal endoscopic surgery the anatomical fidelity of the reconstruction was confirmed; a significant decrease in MD within the chiasma (p<0.0001) was also found. In LHON patients a reduction of MD in the AOP was significantly associated with OCT parameters (p=0.036). Conclusions Multi-shell HARDI diffusion-weighted MRI followed by multi-tissue spherical deconvolution for the DTT reconstruction of the CN I and AOP has been implemented, and its utility demonstrated in clinical practice.

In Vitro And In Vivo Anti-angiogenic Effects Of Hydroxyurea.

Hydroxyurea (HU), or hydroxycarbamide, is used for the treatment of some myeloproliferative and neoplastic diseases, and is currently the only drug approved by the FDA for use in sickle cell disease (SCD). Despite the relative success of HU therapy for SCD, a genetic disorder of the hemoglobin β chain that results in red-cell sickling, hemolysis, vascular inflammation and recurrent vasoocclusion, the exact mechanisms by which HU actuates remain unclear. We hypothesized that HU may modulate endothelial angiogenic processes, with important consequences for vascular inflammation. The effects of HU (50-200 μM; 17-24 h) on endothelial cell functions associated with key steps of angiogenesis were evaluated using human umbilical vein endothelial cell (HUVEC) cultures. Expression profiles of the HIF1A gene and the miRNAs 221 and 222, involved in endothelial function, were also determined in HUVECs following HU administration and the direct in vivo antiangiogenic effects of HU were assessed using a mouse Matrigel-plug neovascularization assay. Following incubation with HU, HUVECs exhibited high cell viability, but displayed a significant 75% inhibition in the rate of capillary-like-structure formation, and significant decreases in proliferative and invasive capacities. Furthermore, HU significantly decreased HIF1A expression, and induced the expression of miRNA 221, while downregulating miRNA 222. In vivo, HU reduced vascular endothelial growth factor (VEGF)-induced vascular development in Matrigel implants over 7 days. Findings indicate that HU is able to inhibit vessel assembly, a crucial angiogenic process, both in vitro and in vivo, and suggest that some of HU's therapeutic effects may occur through novel vascular mechanisms.

The Analgesic Effect Of Dipyrone In Peripheral Tissue Involves Two Different Mechanisms: Neuronal K(atp) Channel Opening And Cb(1) Receptor Activation.

Dipyrone (metamizole) is an analgesic pro-drug used to control moderate pain. It is metabolized in two major bioactive metabolites: 4-methylaminoantipyrine (4-MAA) and 4-aminoantipyrine (4-AA). The aim of this study was to investigate the participation of peripheral CB1 and CB2 cannabinoid receptors activation in the anti-hyperalgesic effect of dipyrone, 4-MAA or 4-AA. PGE2 (100ng/50µL/paw) was locally administered in the hindpaw of male Wistar rats, and the mechanical nociceptive threshold was quantified by electronic von Frey test, before and 3h after its injection. Dipyrone, 4-MAA or 4-AA was administered 30min before the von Frey test. The selective CB1 receptor antagonist AM251, CB2 receptor antagonist AM630, cGMP inhibitor ODQ or KATP channel blocker glibenclamide were administered 30min before dipyrone, 4-MAA or 4-AA. The antisense-ODN against CB1 receptor expression was intrathecally administered once a day during four consecutive days. PGE2-induced mechanical hyperalgesia was inhibited by dipyrone, 4-MAA, and 4-AA in a dose-response manner. AM251 or ODN anti-sense against neuronal CB1 receptor, but not AM630, reversed the anti-hyperalgesic effect mediated by 4-AA, but not by dipyrone or 4-MAA. On the other hand, the anti-hyperalgesic effect of dipyrone or 4-MAA was reversed by glibenclamide or ODQ. These results suggest that the activation of neuronal CB1, but not CB2 receptor, in peripheral tissue is involved in the anti-hyperalgesic effect of 4-aminoantipyrine. In addition, 4-methylaminoantipyrine mediates the anti-hyperalgesic effect by cGMP activation and KATP opening.

Design And Synthesis Of N-acylated Aza-goniothalamin Derivatives And Evaluation Of Their In Vitro And In Vivo Antitumor Activity.

Herein we describe the synthesis of a focused library of compounds based on the structure of goniothalamin (1) and the evaluation of the potential antitumor activity of the compounds. N-Acylation of aza-goniothalamin (2) restored the in vitro antiproliferative activity of this family of compounds. 1-(E)-But-2-enoyl-6-styryl-5,6-dihydropyridin-2(1H)-one (18) displayed enhanced antiproliferative activity. Both goniothalamin (1) and derivative 18 led to reactive oxygen species generation in PC-3 cells, which was probably a signal for caspase-dependent apoptosis. Treatment with derivative 18 promoted Annexin V/7-aminoactinomycin D double staining, which indicated apoptosis, and also led to G2 /M cell-cycle arrest. In vivo studies in Ehrlich ascitic and solid tumor models confirmed the antitumor activity of goniothalamin (1), without signs of toxicity. However, derivative 18 exhibited an unexpectedly lower in vivo antitumor activity, despite the treatments being administered at the same site of inoculation. Contrary to its in vitro profile, aza-goniothalamin (2) inhibited Ehrlich tumor growth, both on the ascitic and solid forms. Our findings highlight the importance of in vivo studies in the search for new candidates for cancer treatment.

Oligomerization, Membrane Association, And In Vivo Phosphorylation Of Sugarcane Udp-glucose Pyrophosphorylase.

Sugarcane is a monocot plant that accumulates sucrose to levels of up to 50% of dry weight in the stalk. The mechanisms that are involved in sucrose accumulation in sugarcane are not well understood, and little is known with regard to factors that control the extent of sucrose storage in the stalks. UDP-glucose pyrophosphorylase (UGPase; EC 2.7.7.9) is an enzyme that produces UDP-glucose, a key precursor for sucrose metabolism and cell wall biosynthesis. The objective of this work was to gain insights into the ScUGPase-1 expression pattern and regulatory mechanisms that control protein activity. ScUGPase-1 expression was negatively correlated with the sucrose content in the internodes during development, and only slight differences in the expression patterns were observed between two cultivars that differ in sucrose content. The intracellular localization of ScUGPase-1 indicated partial membrane association of this soluble protein in both the leaves and internodes. Using a phospho-specific antibody, we observed that ScUGPase-1 was phosphorylated in vivo at the Ser-419 site in the soluble and membrane fractions from the leaves but not from the internodes. The purified recombinant enzyme was kinetically characterized in the direction of UDP-glucose formation, and the enzyme activity was affected by redox modification. Preincubation with H2O2 strongly inhibited this activity, which could be reversed by DTT. Small angle x-ray scattering analysis indicated that the dimer interface is located at the C terminus and provided the first structural model of the dimer of sugarcane UGPase in solution.

Liposomal Lidocaine Gel For Topical Use At The Oral Mucosa: Characterization, In Vitro Assays And In Vivo Anesthetic Efficacy In Humans.

To characterize liposomal-lidocaine formulations for topical use on oral mucosa and to compare their in vitro permeation and in vivo anesthetic efficacy with commercially available lidocaine formulations. Large unilamellar liposomes (400 nm) containing lidocaine were prepared using phosphatidylcholine, cholesterol, and α-tocoferol (4:3:0.07, w:w:w) and were characterized in terms of membrane/water partition coefficient, encapsulation efficiency, size, polydispersity, zeta potential, and in vitro release. In vitro permeation across pig palatal mucosa and in vivo topical anesthetic efficacy on the palatal mucosa in healthy volunteers (double-blinded cross-over, placebo controlled study) were performed. The following formulations were tested: liposome-encapsulated 5% lidocaine (Liposome-Lido5); liposome-encapsulated 2.5% lidocaine (Liposome-Lido2.5); 5% lidocaine ointment (Xylocaina®), and eutectic mixture of lidocaine and prilocaine 2.5% (EMLA®). The Liposome-Lido5 and EMLA showed the best in vitro permeation parameters (flux and permeability coefficient) in comparison with Xylocaina and placebo groups, as well as the best in vivo topical anesthetic efficacy. We successfully developed and characterized a liposome encapsulated 5% lidocaine gel. It could be considered an option to other topical anesthetic agents for oral mucosa.

In Vivo Evaluation Of The Genetic Toxicity Of Rubus Niveus Thunb. (rosaceae) Extract And Initial Screening Of Its Potential Chemoprevention Against Doxorubicin-induced Dna Damage.

Rubus niveus Thunb. plant belongs to Rosaceae family and have been used traditionally to treat wounds, burns, inflammation, dysentery, diarrhea and for curing excessive bleeding during menstrual cycle. The present study was undertaken to investigate the in vivo genotoxicity of Rubus niveus aerial parts extract and its possible chemoprotection on doxorubicin (DXR)-induced DNA damage. In parallel, the main phytochemicals constituents in the extract were determined. The animals were exposed to the extract for 24 and 48h, and the doses selected were 500, 1000 and 2000mg/kg b.w. administered by gavage alone or prior to DXR (30mg/kg b.w.) administered by intraperitoneal injection. The endpoints analyzed were DNA damage in bone marrow and peripheral blood cells assessed by the alkaline alkaline (pH>13) comet assay and bone marrow micronucleus test. The results of chemical analysis of the extract showed the presence of tormentic acid, stigmasterol, quercitinglucoronide (miquelianin) and niga-ichigoside F1 as main compounds. Both cytogenetic endpoints analyzed showed that there were no statistically significant differences (p>0.05) between the negative control and the treated groups with the two higher doses of Rubus niveus extract alone, demonstrating absence of genotoxic and mutagenic effects. Aneugenic/clastogenic effect was observed only at 2000mg/kg dose. On the other hand, in the both assays and all tested doses were observed a significant reduction of DNA damage and chromosomal aberrations in all groups co-treated with DXR and extract compared to those which received only DXR. These results indicate that Rubus niveus aerial parts extract did not revealed any genotoxic effect, but presented some aneugenic/clastogenic effect at higher dose; and suggest that it could be a potential adjuvant against development of second malignant neoplasms caused by the cancer chemotherapic DXR.

In Vivo Determination Of The Volatile Metabolites Of Saprotroph Fungi By Comprehensive Two-dimensional Gas Chromatography.

In this work, we discuss the use of multi-way principal component analysis combined with comprehensive two-dimensional gas chromatography to study the volatile metabolites of the saprophytic fungus Memnoniella sp. isolated in vivo by headspace solid-phase microextraction. This fungus has been identified as having the ability to induce plant resistance against pathogens, possibly through its volatile metabolites. Adequate culture media was inoculated, and its headspace was then sampled with a solid-phase microextraction fiber and chromatographed every 24 h over seven days. The raw chromatogram processing using multi-way principal component analysis allowed the determination of the inoculation period, during which the concentration of volatile metabolites was maximized, as well as the discrimination of the appropriate peaks from the complex culture media background. Several volatile metabolites not previously described in the literature on biocontrol fungi were observed, as well as sesquiterpenes and aliphatic alcohols. These results stress that, due to the complexity of multidimensional chromatographic data, multivariate tools might be mandatory even for apparently trivial tasks, such as the determination of the temporal profile of metabolite production and extinction. However, when compared with conventional gas chromatography, the complex data processing yields a considerable improvement in the information obtained from the samples. This article is protected by copyright. All rights reserved.

Jnk And Ikkβ Phosphorylation Is Reduced By Glucocorticoids In Adipose Tissue From Insulin-resistant Rats.

Peripheral insulin resistance (IR) is one of the main side effects caused by glucocorticoid (GC)-based therapies, and the molecular mechanisms of GC-induced IR are not yet fully elucidated. Thus, we aimed to investigate the effects of dexamethasone treatment on the main components of insulin and inflammatory signaling in the adipose tissue of rats. Male Wistar rats received daily injections of dexamethasone (1mg/kg body weight (b.w.), intraperitoneally (i.p.)) for 5 days (DEX), whereas control rats received saline (CTL). The metabolic status was investigated, and the epididymal fat fragments were collected for lipolysis and western blot analyses. The DEX rats became hyperglycemic, hyperinsulinemic, insulin resistant and glucose intolerant, compared with the CTL rats (P<0.05). The basal glycerol release in the fat fragments was 1.5-fold higher in the DEX rats (P<0.05). The phosphorylation of protein kinase B (PKB) at ser(473) decreased by 44%, whereas, the phosphorylation of insulin receptor substrate (IRS)-1 at ser(307) increased by 93% in the adipose tissue of the DEX rats after an oral bolus of glucose (P<0.05). The basal phosphorylation of c-jun-N-terminal kinase (JNK) and inhibitor of nuclear factor kappa-B (IKKβ) proteins was reduced by 46% and 58%, respectively, in the adipose tissue of the DEX rats (P<0.05). This was paralleled with a significant reduction (47%) in the glucocorticoid receptor (GR) protein content in the adipose tissue of the DEX rats (P<0.05). The insulin-resistant status of rats induced by dexamethasone administration have PKB and IRS-1 activity attenuated in epididymal fat without increases in the phosphorylation of the proinflammatory signals JNK and IKKβ.

Metronidazole-containing gel for the treatment of periodontitis: an in vivo evaluation

The aim of this investigation was to monitor metronidazole concentrations in the gingival crevicular fluid (GCF) collected from periodontal pockets of dogs after treatment with an experimental 15% metronidazole gel. Five dogs had periodontitis induced by cotton ligatures placed subgingivally and maintained for a 30-day period. After the induction period, only pockets with 4 mm or deeper received the gel. Each pocket was filled up to the gingival margin by means of a syringe with a blunt-end needle. GCF was collected in paper strips and quantified in an electronic device before and after 15 minutes, 1 h, 6 h, 24 h and 48 h of gel administration. The GCF samples were assayed for metronidazole content by means of a high performance liquid chromatography method. Concentrations of metronidazole in the GCF of the 5 dogs (mean ± SD, in µg/mL) were 0 ± 0 before gel application and 47,185.75 ± 24,874.35 after 15 minutes, 26,457.34 ± 25,516.91 after 1 h, 24.18 ± 23.11 after 6 h, 3.78 ± 3.45 after 24 h and 3.34 ± 5.54 after 48 h. A single administration of the 15% metronidazole gel released the drug in the GCF of dogs in levels several-fold higher than the minimum inhibitory concentration for some periodontopathogens grown in subgingival biofilms for up to one hour, but metronidazole could be detected in the GCF at least 48 hours after the gel application.

Assessment of the potential genotoxic risk of medicinal Tamarindus indica fruit pulp extract using in vivo assays

Tamarindus indica has been used in folk medicine as an antidiabetic, a digestive aid, and a carminative, among other uses. Currently, there is no information in the toxicology literature concerning the safety of T. indica extract. We evaluated the clastogenic and/or genotoxic potential of fruit pulp extract of this plant in vivo in peripheral blood and liver cells of Wistar rats, using the comet assay, and in bone marrow cells of Swiss mice, using the micronucleus test. The extract was administered by gavage at doses of 1000, 1500 and 2000 mg/kg body weight. Peripheral blood and liver cells from Wistar rats were collected 24 h after treatment, for the comet assay. The micronucleus test was carried out in bone marrow cells from Swiss mice collected 24 h after treatment. The extract made with T. indica was devoid of clastogenic and genotoxic activities in the cells of the rodents, when administered orally at these three acute doses.

Assessment of the Mutagenic Activity of Extracts of Brazilian Propolis in Topical Pharmaceutical Formulations on Mammalian Cells In Vitro and In Vivo

Propolis possesses various biological activities such as antibacterial, antifungal, anti-inflammatory, anesthetic and antioxidant properties. A topically applied product based on Brazilian green propolis was developed for the treatment of burns. For such substance to be used more safely in future clinical applications, the present study evaluated the mutagenic potential of topical formulations supplemented with green propolis extract (1.2, 2.4 and 3.6%) based on the analysis of chromosomal aberrations and of micronuclei. In the in vitro studies, 3-h pulse (G(1) phase of the cell cycle) and continuous (20 h) treatments were performed. In the in vivo assessment, the animals were injured on the back and then submitted to acute (24 h), subacute (7 days) and subchronic (30 days) treatments consisting of daily dermal applications of gels containing different concentrations of propolis. Similar frequencies of chromosomal aberrations were observed for cultures submitted to 3-h pulse and continuous treatment with gels containing different propolis concentrations and cultures not submitted to any treatment. However, in the continuous treatment cultures treated with the 3.6% propolis gel presented significantly lower mitotic indices than the negative control. No statistically significant differences in the frequencies of micronuclei were observed between animals treated with gels containing different concentrations of propolis and the negative control for the three treatment times. Under the present conditions, topical formulations containing different concentrations of green propolis used for the treatment of burns showed no mutagenic effect in either test system, but 3.6% propolis gel was found to be cytotoxic in the in vitro test.