1,721,213 research outputs found
Effects of acetyl-L-carnitine on the survival of adult rat sensory neurons in primary cultures
No full textAcetyl-L-carnitine produces a significant increase in the survival time-course of adult rat sensory neurons maintained in primary cultures up to 40 days. The analysis of our data suggests that 200 microM acetyl-L-carnitine added to the medium, slows down neuronal decay especially in the first 10 days in vitro, sparing a fraction of cells which would otherwise be lost. Patch-clamp recordings from these neurons show that superfusion with acetyl-L-carnitine (100-1000 microM) does not induce any membrane current. In addition an agonist muscarinic effect particularly concerning high-voltage activated calcium channel modulation appears to be ruled out. In conclusion our data favour the role of acetyl-L-carnitine in the trophism of sensory neurons in adult rats. In agreement with other in vivo experiments our data reinforce the hypothesis that this substance might be involved in reducing neuronal loss observed in nervous system aging
ADIPOSE-DERIVED STEM CELLS (ASCS) FOR FUTURE CELLULAR THERAPIES IN MUSCLE-SKELETAL TISSUES REGENERATION
Full text linkEvery year several patients have to deal with bone tissue loss due to trauma or diseases. Bone tissue engineering aims to restore or repair musculoskeletal disorders through the development of bio-substitutes that require the use of cells and scaffolds which should possess both adequate mechanical properties and interconnecting pores to allow cellular infiltration, graft integration and vascularization. The ideal cell for tissue engineering should possess a potential plasticity with the ability to functionally repair the damaged tissue, and it should be available in large amount. Mesenchymal stem cells (MSCs) are present in many adult tissues, and adipose tissue represents an attractive source of MSCs for researchers and clinicians of nearly all medical specialties. Adipose-derived stem cells (ASCs) are similar to MSCs isolated from bone marrow, placenta, and umbilical cord blood in morphology, immunophenotype, and differentiation ability, and they represent a promising approach of bone regeneration. Additional features of ASCs are their immunoregolatory and anti-inflammatory properties both in vivo and in vitro and their low immunogenicity.
Since several years our laboratory is studying mesenchymal stem cells isolated from human and animal adipose tissues. Human ASCs (hASCs) have been characterized by their immunophenotype, their self-renewal potential, and they have been induced to differentiate towards adipogenic, osteogenic and chondrogenic lineages. The ability of hASCs to grow in the presence of several scaffolds has also been tested. hASCs adhered to the surface of tested biomaterials, filling the pores and forming a 3D web-like structure, allowing these progenitor cells to osteo-differentiate more efficiently respect to cells maintained on polystyrene. Since our interest was to regenerate muscle-skeletal defects by ASCs in pre-clinical models, we first studied ASCs isolated from adipose tissue of rat (rASCs), rabbit (rbASCs) and pig (pASCs), considered good models in the orthopaedic field. We have shown that animal ASCs behaved similarly to the human ones, and, in collaboration with the Faculty of Veterinary Medicine of University of Milan and the IRCCS Galeazzi Orthopaedic Institute of Milan, we have tested the ability of autologous ASCs to regenerate a full-thickness critical-size bone defect in rabbits. The experimental study was conducted on the tibiae of 12 New Zealand rabbits, and from 6 rabbits out of 12 we have collected adipose tissue from the interscapular region. We have isolated 2.8x105±1.9x105 rbASCs per ml of raw tissue, and after 3-4 days in culture the cells showed the typical fibroblast-like morphology. One week later, all the 6 cellular populations started to steadily proliferate, and they generated fibroblast (CFU-F) and osteoblast (CFU-O) colonies, highlighting the presence of osteogenic progenitors. Indeed, when rbASCs were induced to osteo-differentiate, either after 7 and 14 days, we have observed an up-regulation of specific osteogenic markers, such as alkaline phosphatase (ALP, +28.9%), collagen (+105.9%) and extracellular calcified matrix (+168.1%), compared to undifferentiated cells. In parallel, testing HA, the scaffold selected for the in vivo experiment, we found that rbASCs were osteoinduced; indeed the presence of HA granules increased per se the amount of collagen production (+48.2%).
1.5x106 undifferentiated rbASCs were seeded on custom-made HA disks (8 mm Ø x 4 mm ↕), and the day after, each bioconstruct was implanted into the lesion created in the tibia of each rabbit. We had an additional experimental group of defects where the same number of rbASCs were inserted in the lesion as a semi-liquid suspension; moreover, as controls, we treated 6 lesioned tibia with just the scaffolds, and we left 6 untreated lesioned bone.
8 weeks after surgery animals were sacrificed and the tibia explanted. A macroscopic analysis showed no bone resorption, no abnormal bone callous formation, no fractures, infection or inflammatory reactions, and all the bone defects were completely filled without any significant differences among the four groups.
Interestingly, in the presence of scaffold seeded with rbASCs, histology and immunohistochemistry showed a new bone tissue more mature and similar to the native bone. These data have also been confirmed by biomechanical tests: indeed, the mechanical properties of the bone defect treated by rbASCs-HA were improved, suggesting that these constructs bore mechanical loading with an increase in stiffness of 19.8% and in hardness of 31.6% respect to just HA treated group, indicating that the bioconstructs made out of autologous rbASCs and hydroxyapatite might ameliorate the treatment for large bone defects. We would suggest the use of ASCs as a safe cellular therapy in future clinical applications where a large bone defect needs to be treated. These promising results on small size animals allow us to plan a new study on large size animals such as minipigs.
However, before moving to the clinic, we know that there are several important aspects that need to be faced regarding safeness and the features of the candidate patients:
1. may the “quality” of hASCs be affected by the donor’s physiological or pathological conditions?
2. may the use of pharmacological treatment enhance cellular plasticity of multipotent cells?
3. may the use of immunoselected hASCs ameliorate tissue regeneration in the field of muscle-skeletal?
We have addressed some of these aspects, comparing different populations of hASCs from subcutaneous adipose tissue of healthy-young-female donors (hASCs45 y/o n=14, mean age 56±7 years, mean BMI=28.4±1.8). The cellular yield of hASCs derived from older donors was 2.5 fold greater than the one of hASCs<35 y/o, whereas hASCs from younger donors were more clonogenic than hASCs isolated from older ones, with an increase of 129%. No significant differences were observed looking at their immunophenotype.
When hASCs were induced to differentiate into cells of the adipogenic and osteogenic lineages, the donor’s age did not affect their adipogenic differentiation, whereas the osteogenic one was significantly affected by age both in the absence and in the presence of three-dimensional scaffolds, showing a decreased ALP basal levels of about 10-fold in hASCs>45 y/o respect to hASCs<35 y/o.
These results seems to indicate that ASCs from different donors could behave differently.
Trying to overcome this aspect we have used different approaches, and we have studied if Reversine, a synthetic purine already known to increase plasticity of terminally differentiated cells, might improve the differentiation ability of hASCs. 72 hours treatment with 50 nM Reversine induced hASCs to differentiate into osteoblast like-cells (+45% of alkaline phosphatase activity), smooth muscle cells (+89% of α-actin expression) and skeletal muscle cells (myotubes formation) compared to control hASCs.
Moreover, since it is known that CD34 and L-NGFR positive cells define a subset of high proliferative and multipotent MSCs, we have immunoselected, these progenitor cells from hASC populations. In contrast to the whole population, the immunoseparated fractions maintained their undifferentiate state and their ability to differentiate much longer during culture. We have shown that both CD34+ and L-NGFR+ hASCs can be used as alternative candidates for tissue engineering and regenerative medicine applications. In particular, due to the improved ability of L-NGFR positive cells to adipo- and chondro-differentiate, they appear an ideal tool in reconstructive plastic surgery and cartilage regeneration.
From our data, and the ones from researchers in other fields, we believe that in the near future adipose-derived stem cells might be considered a safe tool in regenerative medicine. Furthermore, to improve this “cellular therapy”, we could either pre-treat ASCs with molecules, such as drugs and/or siRNAs known to affect specific differentiation pathways, or by selecting subpopulations of progenitor cells which may be used as allogenic implants. Next step will be to confirm our in vivo data in a large size animal model such as minipig, and then to test if pre-treated cells or selected population might be used in an autologous and allogenic small size animal model
L’encefalina inibisce l’influsso di Ca2+ attraverso i canali del Ca2+ voltaggio-dipendenti in neuroni sensoriali periferici e centrali di mammifero.
No full textRIASSUNTO E' stato studiato l'effetto della D-ala2-D-leu5-encefalina (DADLE) 400 nM sulle correnti di calcio HVA (High Voltage Activated) e LVA (Low Voltage Activated). Gli esperimenti sono stati condotti su neuroni dei gangli delle radici dorsali e del talamo ventrobasale di ratto con la tecnica del patch-clamp whole-cell. La DADLE inibiva le correnti di Ca2+ HVA sia dei neuroni periferici (-59% +/-2.47 SEM n = 39 su 43 registrati) che centrali (-67% +/-3.2 n = 22 su 27), mentre solo nei neuroni talamici le correnti di Ca2+ LVA erano sensibili all' effetto della DADLE (-40% +/-3.3 n = 4 su 11). Nei neuroni periferici si potevano distinguere due tipi di modulazione: una dipendente e l'altra indipendente dal voltaggio. I risultati suggeriscono due distinti ruoli funzionali della inibizione esercitata dalla DADLE sulle correnti di Ca2+ HVA dei neuroni periferici. La DADLE potrebbe esercitare inoltre un ruolo significativo nel controllo della scarica ritmica dei neuroni di relè talamici
Low-voltage activated calcium channels are differently affected by nimodipine
No full textVoltage-dependent Ca2+ channels in adult rat sensory neurones were studied as far as their characterization and nimodipine effects are concerned, using patch-clamp whole-cell technique. Low-voltage activated (LVA) Ca2+ currents were identified according to activation and inactivation kinetics and sensitivity to amiloride. Nimodipine (10 nM) caused a decrease in LVA Ca2+ current amplitude (-40% +/- 2.2 s.e.m., n = 11 out of 30 with LVA Ca2+ currents). Conversely, in 6 neurones out of 30 nimodipine increased the Ca2+ current amplitude (+ 10% +/- 2). In some unaffected neurones (n = 5) nimodipine was also tested at higher concentrations (up to 5 microM) without any appreciable effect. In conclusion, nimodipine was partly able to block LVA calcium channels even at nanomolar concentrations. Supposing nimodipine acts directly on the channel, it can be assumed that there may be different types of LVA calcium channels in sensory neurones
ANTIFERROMAGNETISM OF CUO2 LAYERS WITHIN A SLAVE-BOSON APPROACH
No full textThe antiferromagnetic ground state of CuO2 layers is studied at the mean-field level within a slave-boson approach to a multiband Hubbard model which includes the dx2-y2 orbital for Cu and the px and py orbitals for O. The stability of the antiferromagnetism is tested by varying the separation between p- and d-hole energy levels and by allowing direct O-O hopping. Results for the effective exchange integral, the antiferromagnetic gap, the percentage of d character of holes at the Fermi level, and the Cu magnetic moment are discussed by varying. © 1990 The American Physical Society
Rabbit Adipose-derived Stem Cells and tibia repair
No full textAdipose-derived stem cells (ASCs) may represent, alone or in combination with different scaffolds, a novel and efficient approach for bone regeneration. Here, we describe how autologous rabbit ASCs (rbASCs) isolated from interscapular adipose tissue, expanded and characterized in vitro, are used to regenerate a full-thickness bone defect in the tibial crest of New Zealand rabbits.
The animals have been divided in 4 groups: one group where the lesions have been treated with rbASCs seeded on hydroxyapatite-disk (rbASCs-HA), one group with only rbASCs, one with just HA, and one untreated group (just defect). The follow-up was of eight weeks.
Meanwhile, rbASCs have been characterized in vitro: these progenitor cells show a homogenous high proliferation rate and a marked clonogenic ability. Moreover, rbASCs demonstrate an osteogenic potential that has been evaluated by the expression of specific bone markers such as alkaline phosphatase, collagen, osteonectin and extracellular calcified matrix deposition, both in the absence and in the presence of hydroxyapatite.
Eight weeks after surgical interventions, gross appearance, X-rays, histological analyses and biomechanical tests were performed on all the animals.
The macroscopic analyses of all the tibias show a satisfactory filling of the lesions without any significant difference in terms of stiffness. By X-rays, a good osteo-integration appears in both scaffold-treated groups; despite the fact that HA was not completely resorbed, cells-HA treated bones show a more efficient scaffold resorption than the other group. In addition, the scaffold-treated defects show a better bone formation compared to the control samples. In particular, the new bone, formed in the presence of rbASCs-HA, is more mature and similar to the native one showing an improvement in bone mechanical properties.
These results indicate that autologous ASCs-hydroxyapatite bio-construct may be a potential treatment for the regeneration of bone defects
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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