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2.
PLoS One ; 12(5): e0178182, 2017.
Article in English | MEDLINE | ID: mdl-28542481

ABSTRACT

Hyperbaric oxygen therapy (HBOT) is a noninvasive widely applied treatment that increases the oxygen pressure in tissues. In cochlear implant (CI) research, intracochlear application of neurotrophic factors (NTFs) is able to improve survival of spiral ganglion neurons (SGN) after deafness. Cell-based delivery of NTFs such as brain-derived neurotrophic factor (BDNF) may be realized by cell-coating of the surface of the CI electrode. Human mesenchymal stem cells (MSC) secrete a variety of different neurotrophic factors and may be used for the development of a biohybrid electrode in order to release endogenously-derived neuroprotective factors for the protection of residual SGN and for a guided outgrowth of dendrites in the direction of the CI electrode. HBOT could be used to influence cell behaviour after transplantation to the inner ear. The aim of this study was to investigate the effect of HBOT on the proliferation, BDNF-release and secretion of neuroprotective factors. Thus, model cells (an immortalized fibroblast cell line (NIH3T3)-native and genetically modified) and MSCs were repeatedly (3 x - 10 x) exposed to 100% oxygen at different pressures. The effects of HBO on cell proliferation were investigated in relation to normoxic and normobaric conditions (NOR). Moreover, the neuroprotective and neuroregenerative effects of HBO-treated cells were analysed by cultivation of SGN in conditioned medium. Both, the genetically modified NIH3T3/BDNF and native NIH3T3 fibroblasts, showed a highly significant increased proliferation after five days of HBOT in comparison to normoxic controls. By contrast, the number of MSCs was decreased in MSCs treated with 2.0 bar of HBO. Treating SGN cultures with supernatants of fibroblasts and MSCs significantly increased the survival rate of SGN. HBO treatment did not influence (increase / reduce) this effect. Secretome analysis showed that HBO treatment altered the protein expression pattern in MSCs.


Subject(s)
Brain-Derived Neurotrophic Factor/metabolism , Hyperbaric Oxygenation , Neuroprotection/physiology , Animals , Brain-Derived Neurotrophic Factor/genetics , Cell Proliferation/physiology , Cell Survival/physiology , Cell- and Tissue-Based Therapy , Culture Media, Conditioned , Female , Gene Expression Profiling , Humans , Male , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells/metabolism , Mice , NIH 3T3 Cells/metabolism , NIH 3T3 Cells/transplantation , Nerve Regeneration/physiology , Neuronal Outgrowth/physiology , Neurons/cytology , Neurons/physiology , Spiral Ganglion/cytology , Spiral Ganglion/physiology , Young Adult
3.
PLoS One ; 8(12): e80490, 2013.
Article in English | MEDLINE | ID: mdl-24349001

ABSTRACT

The formation of gliosis around implant electrodes for deep brain stimulation impairs electrode-tissue interaction. Unspecific growth of glial tissue around the electrodes can be hindered by altering physicochemical material properties. However, in vitro screening of neural tissue-material interaction requires an adequate cell culture system. No adequate model for cells dissociated from the inferior colliculus (IC) has been described and was thus the aim of this study. Therefore, IC were isolated from neonatal rats (P3_5) and a dissociated cell culture was established. In screening experiments using four dissociation methods (Neural Tissue Dissociation Kit [NTDK] T, NTDK P; NTDK PN, and a validated protocol for the dissociation of spiral ganglion neurons [SGN]), the optimal media, and seeding densities were identified. Thereafter, a dissociation protocol containing only the proteolytic enzymes of interest (trypsin or papain) was tested. For analysis, cells were fixed and immunolabeled using glial- and neuron-specific antibodies. Adhesion and survival of dissociated neurons and glial cells isolated from the IC were demonstrated in all experimental settings. Hence, preservation of type-specific cytoarchitecture with sufficient neuronal networks only occurred in cultures dissociated with NTDK P, NTDK PN, and fresh prepared papain solution. However, cultures obtained after dissociation with papain, seeded at a density of 2×10(4) cells/well and cultivated with Neuro Medium for 6 days reliably revealed the highest neuronal yield with excellent cytoarchitecture of neurons and glial cells. The herein described dissociated culture can be utilized as in vitro model to screen interactions between cells of the IC and surface modifications of the electrode.


Subject(s)
Neuroglia/cytology , Neurons/cytology , Papain/metabolism , Animals , Microscopy, Atomic Force , Oligodendroglia/cytology , Rats , Rats, Sprague-Dawley
4.
Neuropharmacology ; 75: 416-25, 2013 Dec.
Article in English | MEDLINE | ID: mdl-23973291

ABSTRACT

Activins are regulators of embryogenesis, osteogenesis, hormones and neuronal survival. Even though activin receptor type II has been detected in spiral ganglion neurons (SGN), little is known about the role of activins in the inner ear. An activin-mediated neuroprotection is of considerable clinical interest since SGN are targets of electrical stimulation with cochlear implants in hearing impaired patients. Thus, the presence of activin type-I and type-II receptors was demonstrated immunocytochemically and the individual and combined effects of activin A, erythropoietin (EPO) and brain-derived neurotrophic factor (BDNF) on SGN were examined in vitro. SGN isolated from neonatal rats (P 3-5) were cultured in serum-free medium supplemented with activin A, BDNF and EPO. Compared to the negative control, survival rates of SGN were significantly improved when cultivated individually with activin A (p<0.001) and in combination with BDNF (p<0.001). Neither neurite outgrowth nor neuronal survival was influenced by the addition of EPO to activin A-treated neurons. However, when all three factors were added, a significantly (p<0.001) improved neuronal survival was observed (61.2±3.6%) compared to activin A (25.4±2.1%), BDNF (22.8±3.3%) and BDNF+EPO (19.2±1.5%). Under the influence of the EPO-inhibitors, this increase in neuronal survival was blocked. Acting with BDNF and EPO to promote neuronal survival in vitro, activin A presents an interesting factor for pharmacological intervention in the inner ear. The present study demonstrates a synergetic effect of a combined therapy with several trophic factors.


Subject(s)
Activins/pharmacology , Brain-Derived Neurotrophic Factor/pharmacology , Erythropoietin/pharmacology , Neurons/drug effects , Spiral Ganglion/cytology , Animals , Animals, Newborn , Cell Survival/drug effects , Cells, Cultured , Culture Media, Serum-Free/pharmacology , Dose-Response Relationship, Drug , Drug Combinations , Female , Follistatin/metabolism , Male , Nerve Tissue Proteins/metabolism , Rats , Rats, Sprague-Dawley
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