Mesenchymal Stem-Cell Remodeling of Adsorbed Type-I Collagen-The Effect of Collagen Oxidation.

This study describes the effect of collagen type I (Col I) oxidation on its physiological remodeling by adipose tissue-derived mesenchymal stem cells (ADMSCs), both mechanical and proteolytic, as an in vitro model for the acute oxidative stress that may occur in vivo upon distinct environmental changes. Morphologically, remodeling was interpreted as the mechanical rearrangement of adsorbed FITC-labelled Col I into a fibril-like pattern. This process was strongly abrogated in cells cultured on oxidized Col I albeit without visible changes in cell morphology. Proteolytic activity was quantified utilizing fluorescence de-quenching (FRET effect). The presence of ADMSCs caused a significant increase in native FITC-Col I fluorescence, which was almost absent in the oxidized samples. Parallel studies in a cell-free system confirmed the enzymatic de-quenching of native FITC-Col I by Clostridial collagenase with statistically significant inhibition occurring in the oxidized samples. Structural changes to the oxidized Col I were further studied by differential scanning calorimetry. In the oxidized samples, an additional endotherm with sustained enthalpy (∆H) was observed at 33.6 °C along with Col I's typical one at 40.5 °C. Collectively, these data support that the remodeling of Col I by ADMSCs is altered upon oxidation due to intrinsic changes to the protein's structure, which represents a novel mechanism for the control of stem cell behavior.

Antioxidant effect of MnTE-2-PyP on lung in asthma mice model.

AIM: To investigate the effects of MnTE-2-PyP on some markers of antioxidant defence system in asthma mice model. MATERIAL AND METHODS: The animals were divided into four groups: group 1, controls; group 2, injected with ovalbumin, group 3, treated with MnTE-2-PyP, and group 4, treated with ovalbumin and MnTE-2-PyP. The activities of superoxide dismutase, catalase, glutathione peroxidase and nonprotein sulfhydryl groups content (NPSH) were determined in lung homogenate. RESULTS: The activities of superoxide dismutase and catalase in group 2 decreased significantly as compared to control group. The decrease of the same enzymes in group 4 was lower and significant as compared to group 2. Changes in the glutathione peroxidase activity showed a similar dynamics. The NPSH groups content decreased in group 2. In group 4 this decrease was relatively lower as compared to group 2. CONCLUSIONS: The application of MnTE-2-PyP mitigated the effects of oxidative stress in asthma mice model.

Morphological and Quantitative Evidence for Altered Mesenchymal Stem Cell Remodeling of Collagen in an Oxidative Environment-Peculiar Effect of Epigallocatechin-3-Gallate.

Mesenchymal stem cells (MSCs) are involved in the process of extracellular matrix (ECM) remodeling where collagens play a pivotal role. We recently demonstrated that the remodeling of adsorbed collagen type I might be disordered upon oxidation following its fate in the presence of human adipose-derived MSC (ADMSCs). With the present study we intended to learn more about the effect of polyphenolic antioxidant Epigallocatechin-3-gallate (EGCG), attempting to mimic the conditions of oxidative stress in vivo and its putative prevention by antioxidants. Collagen Type I was isolated from mouse tail tendon (MTC) and labelled with FITC before being oxidized according to Fe2+/H2O2 protocol. FITC-collagen remodeling by ADMSC was assessed morphologically before and after EGCG pretreatment and confirmed via detailed morphometric analysis measuring the anisotropy index (AI) and fluorescence intensity (FI) in selected regions of interest (ROI), namely: outside the cells, over the cells, and central (nuclear/perinuclear) region, whereas the pericellular proteolytic activity was measured by de-quenching fluorescent collagen probes (FRET effect). Here we provide morphological evidence that MTC undergoes significant reorganization by the adhering ADMSC and is accompanied by a substantial activation of pericellular proteolysis, and further confirm that both processes are suppressed upon collagen oxidation. An important observation was that this abrogated remodeling cannot be prevented by the EGCG pretreatment. Conversely, the detailed morphometric analysis showed that oxidized FITC-collagen tends to accumulate beneath cells and around cell nuclei, suggesting the activation of alternative routes for its removal, such as internalization and/or transcytosis. Morphometric analysis also revealed that both processes are supported by EGCG pretreatment.

Activation-dependent descending reflex evacuation motority of anal canal in rat model.

The evacuative motor responses of the anal canal and recto-anal reflexes during defecation were studied in an isolated rat recto-anal model preparation using (i) partitioned organ bath, (ii) electrical stimulation, (iii) balloon distension and (iv) morphological techniques. Electrical field stimulation applied to the anal canal or to the distal part of the rectum elicited tetrodotoxin (10(-7) M)-sensitive frequency-dependent local or descending contractions of the anal canal and the local responses were bigger in amplitude (14.9 ± 1.35 mN) than the descending contractions (5.3 ± 0.7 mN at frequency of 5 Hz, p < 0.05). The balloon-induced distension of the distal rectum evoked descending responses of the anal canal consisting of a short contraction (1.50 ± 0.18 mN) followed by deep relaxation (3.12 ± 0.34 mN). In the presence of atropine (3 x 10(-7) M) the electrically-elicited (5 Hz) local or descending contractions of the anal canal were suppressed and a relaxation revealed. The initial contraction component of the distension-induced response was decreased while the relaxation was not changed. During atropine treatment, spantide (10(-7) M) lowered even more the contractile component of the anal canal response. NG-nitro-L-arginine (5 x 10(-4) M) enhanced the contraction, prevented the atropine-dependent relaxation of the electrically-elicited response and inhibited the distension-induced relaxation. L-Arginine (5 x 10(-4) M) suppressed the contraction and extended the relaxation. ChAT-, substance P- and NADPH-diaphorase-positive perikarya and nerve fibers were observed in myenteric ganglia of the anal canal. The results suggest activation-dependent descending reflex motority of the anal canal involving electrical stimulation-displayed cholinergic and tachykininergic and distension manifested nitrergic neuro-muscular communications.

Subchronic Central Administration of Cannabinoid Ligands Modulates Nociception in Bulbectomized Rats.

INTRODUCTION: Endocannabinoid system is involved in neuropsychiatric disorders such as major depression. The bilaterally olfactory bulbectomized rat is widely used as an animal model of depression. The removal of the olfactory bulbs produces behavioural, physiological, and neurochemical alterations resembling clinical depression. There is increasing evidence that highlights the important role of cannabinoid signalling in depression and nociception. AIM: To investigate the effect of CB1 receptor agonist HU 210 and CB1 receptor antagonist SR 141716A administered icv subchronically (for 7 days) on nociception of rats with model of depression - bilateral olfactory bulbectomy (OBX). MATERIAL AND METHODS: Experimental model of depression - bilateral olfactory bulbectomy (OBX). Bilaterally olfactory bulbectomized rats were used as an experimental model of depression. HU 210 (5 µg) or SR 141716A (3 µg) were infused icv for 7 consecutive days, starting 15 days after the olfactory bulbectomy. Nociception was examined by applying paw pressure test (analgesy-meter) evaluating the rat pain threshold. On day 7, five minutes after the last microinjection, the rats were tested in an analgesy-meter and their mechanically evoked pain responses were measured in arbitrary units (AU). RESULTS: Microinjections of HU 210 (5 µg) significantly decreased the pain threshold in olfactory bulbectomized rats, while SR 141716A (3 µg) exerted antinociceptive effect by increasing the pain threshold. CONCLUSIONS: Data point to an involvement of CB1 receptors in depression-like behaviour and nociception in olfactory bulbectomized rats and support the data for the association between depressive disorder and pain pathways.

Effect of Aronia melanocarpa fruit juice on amiodarone-induced pneumotoxicity in rats.

BACKGROUND: The fruits of Aronia melanocarpa (Michx.) Elliot is extremely rich in biologically active polyphenols. OBJECTIVE: We studied the protective effect of A. melanocarpa fruit juice (AMFJ) in a model of amiodarone (AD)-induced pneumotoxicity in rats. MATERIALS AND METHODS: AD was instilled intratracheally on days 0 and 2 (6.25 mg/kg). AMFJ (5 mL/kg and 10 mL/kg) was given orally from day 1 to days 2, 4, 9, and 10 to rats, which were sacrificed respectively on days 3, 5, 10, and 28 when biochemical, cytological, and immunological assays were performed. RESULTS: AMFJ antagonized AD-induced increase of the lung weight coefficient. In bronchoalveolar lavage fluid, AD increased significantly the protein content, total cell count, polymorphonuclear cells, lymphocytes and the activity of lactate dehydrogenase, acid phosphatase and alkaline phosphatase on days 3 and 5. In AMFJ-treated rats these indices of direct toxic damage did not differ significantly from the control values. In lung tissue, AD induced oxidative stress measured by malondialdehyde content and fibrosis assessed by the hydroxyproline level. AMFJ prevented these effects of AD. In rat serum, AD caused a significant elevation of interleukin IL-6 on days 3 and 5, and a decrease of IL-10 on day 3. In AMFJ-treated rats, these indices of inflammation had values that did not differ significantly from the control ones. CONCLUSION: AMFJ could have a protective effect against AD-induced pulmonary toxicity as evidenced by the reduced signs of AD-induced direct toxic damage, oxidative stress, inflammation, and fibrosis.

Region-related modular nerve-dependent motor activity in anorectum--cholinergic and nitrergic contribution to rat model.

Disturbances of enteric nerve-mediated anorectal evacuation mechanisms have medical and social impact. The study aimed at further eliciting the contribution of cholinergic and nitrergic neurotransmission systems to modular nerve networks in different regions of Wistar rat anorectum. Electrical field stimulation (EFS, 0.8 ms, 40 V, 2, 5 or 10 Hz, 20 s), computerized mechanographic on-line setup and drugs were used to evaluate the motor responses of isolated rings from circular muscle of rectum (proximal, middle, and distal part), internal anal sphincter, and anal canal. Twitch-like frequency-dependent contractions, more pronounced in rectal preparations, characterized the modular motor responses of rectal circular muscle rings and anal canal. Depending on the frequency of stimulation, the motor activity of internal anal sphincter varied from deep long-lasting relaxation to initial short-lasting relaxation, followed by a contraction. Electrically-evoked responses of anorectal preparations were tetrodotoxin (0.1 microM)-sensitive. In the presence of atropine (0.3 microM) the contractions of rectal rings decreased, relaxation of internal anal sphincter increased and inhibition of the contractions of the anal canal occurred, followed by relaxation. During atropine treatment, NG-nitro-L-arginine (0.5 microM) increased the contractile responses and suppressed internal anal sphincter relaxations. L-arginine (0.5 microM) decreased the contractions and extended the relaxations of internal anal sphincter and anal canal. Our results suggest that cholinergic and nitrergic systems are not equally involved in modular nerve networks of various regions of anorectum. Cholinergic transmission is more expressed in distal rectum, underlying its contractile potency, while nitric oxide-dependent transmission(s) control the relaxation ability of the internal anal sphincter and anal canal.

Effects of MnTnHex-2-PyP on lung antioxidant defence system in asthma mice model.

We aimed to study the MnTnHex-2-PyP effect on some markers of lung antioxidant defence system in mice asthma model.The study was carried out on 28 C57B1/6 mice divided into four treatment groups: group 1 - controls; group 2 - injected and inhaled with ovalbumin; group 3 - treated with MnTnHex-2-PyP and inhaled with phosphate buffered saline; group 4 - injected with ovalbumin and MnTnHex-2-PyP but also inhaled with ovalbumin. On days 24, 25 and 26, mice from groups 1 and 2 were inhaled with PBS for 30 min, and those from groups 2 and 4 were given a 1% ovalbumin solution. One hour before inhalation, and 12 hours later the animals from groups 1 and 2 were injected i.p. with 100 µl PBS, and those from groups 3 and 4 received a 100 µl MnTnHex-2-PyP solution in PBS, сontaining 0,05mg/kg. The animals were killed by exsanguination 48 hours after the last inhalation for obtaining a lung homogenate. The activities of superoxide dismutase, catalase, glutathione peroxidase and the non-protein sulphhydryl group content in the lung homogenate were investigated. Ovalbumin decreased the activities of superoxide dismutase (p=0.01), catalase (p=0.002), glutathione peroxidase and non-protein sulphhydryl groups content (p<0.001) in comparison to controls. In group 4 (ovalbumin and MnTnHex-2-PyP) the activities of superoxide dismutase (p=0.044), catalase (p=0.045), glutathione peroxidase (p=0.002), and the non-protein sulphhydryl groups content (p<0.001) were significantly increased compared to ovalbumin (group 2).MnTnHex-2-PyP restored the activities of basic enzymes in the lung antioxidant defence system in ovalbumin-induced asthma mice model, 48 hours after the last nebulization.