RESUMEN
The focus of cell replacement therapies (CRTs) for Parkinson's disease has been on delivering dopamine-producing cells to the striatum. Fetal grafts have proven the feasibility of this approach, but an appropriate source of replacement cells has restricted the clinical translation. Bone marrow stromal cells (BMSCs) have been heralded as an ideal source of dopaminergic (DAergic) replacement cells, as they are viewed as ethically acceptable, easily procured, and readily expanded. It is known that they confer functional benefits, particularly in stroke models, through the release of neurotrophic factors, but their transdifferentiation into neurons is still under contention. We sought to evaluate the neuronal phenotype and functional capacity of adult rat BMSCs after exposure to a novel multistep in vitro differentiation protocol compared with cells exposed to other reported neuronal differentiation conditions. We employed a systematic, comprehensive method of assessment to determine the neuronal differentiation capacity of BMSCs. Our fluorescence-activated cell sorting, immunofluorescent and semiquantitative polymerase chain reaction results confirmed that undifferentiated BMSCs isolated based on their adherence to plastic are of mesenchymal origin and express a range of lineage markers. After exposure to preinduction and neuronal induction steps, BMSCs down-regulate markers of other lineages but fail, as assessed by patch clamp, to differentiate into functional neurons. Thus, for BMSCs to be considered a source of DAergic neuronal replacement cells, their ability to transdifferentiate terminally along a neuronal lineage first must be clarified before attempting to direct more complex specification process required for them to be used in Parkinson's-disease-focused CRTs.
Asunto(s)
Células de la Médula Ósea , Trasplante de Médula Ósea , Neuronas/fisiología , Enfermedad de Parkinson/terapia , Células del Estroma/trasplante , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Diferenciación Celular , Linaje de la Célula , Femenino , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Células Madre Mesenquimatosas/citología , Neuronas/citología , Neuronas/metabolismo , Fenotipo , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células del Estroma/citología , Células del Estroma/metabolismoRESUMEN
PURPOSE: After partial optic nerve (ON) injury, intact retinal ganglion cells (RGCs) undergo secondary death, but the topographic distribution of this death is unknown, and it is unclear which cell death pathways are involved. Although the calcium channel blocker lomerizine reduces RGC death after partial ON injury, it is unknown whether this drug alleviates necrotic or apoptotic death. METHODS: The dorsal ON was transected in adult Piebald-Virol-Glaxo (PVG) rats, and the site of secondary RGC death was determined using anterograde and retrograde DiI tracing. RGC death was assessed at 2 and 3 weeks. Retrograde tracing with fluorogold injected into the superior colliculus 3 days before euthanatization was used to identify RGCs undergoing secondary death. Overall cell loss was quantified using betaIII-tubulin immunohistochemistry. Lomerizine (30 mg/kg, oral) or vehicle was given twice daily, and retinal wholemounts were analyzed for necrotic morphology (nucleic acid stain) or anticleaved caspase-3 expression at 2 and 3 weeks. RESULTS: Ventral retina was identified as the site of secondary RGC death, and central and dorsal retinae were defined as sites of both primary and secondary death. Overall RGC loss occurred by 2 weeks in central and ventral retina (P < 0.05) and by 3 weeks in dorsal retina (P < 0.05). Secondary RGC death was characterized mainly by necrotic morphology, with caspase-3 expression in some RGCs. Lomerizine reduced secondary necrosis at 2 weeks and secondary caspase-3 expression at 3 weeks. CONCLUSIONS: Lomerizine had differential effects on necrotic and apoptotic death with time, but its inability to completely prevent secondary death suggests that full neuroprotection will require combinatorial treatments.
Asunto(s)
Bloqueadores de los Canales de Calcio/farmacología , Fármacos Neuroprotectores/farmacología , Piperazinas/farmacología , Enfermedades de la Retina/prevención & control , Células Ganglionares de la Retina/efectos de los fármacos , Células Ganglionares de la Retina/patología , Animales , Carbocianinas , Caspasa 3/metabolismo , Recuento de Células , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Colorantes Fluorescentes , Traumatismos del Nervio Óptico/complicaciones , Ratas , Enfermedades de la Retina/etiología , Enfermedades de la Retina/patología , Células Ganglionares de la Retina/metabolismo , Tubulina (Proteína)/metabolismoRESUMEN
Secondary degeneration is a form of 'bystander' damage that can affect neural tissue both nearby and remote from an initial injury. Partial optic nerve transection is an excellent model in which to unequivocally differentiate events occurring during secondary degeneration from those resulting from primary CNS injury. We analysed the primary injury site within the optic nerve (ON) and intact areas vulnerable to secondary degeneration. Areas affected by the primary injury showed morphological disruption, loss of beta-III tubulin axonal staining, reduced myelinated axon density, greater proteoglycan expression (phosphacan), increased microglia and macrophage numbers and increased oxidative stress. Similar, but less extreme, changes were seen in areas of the optic nerve undergoing secondary degeneration. The CNS-specific L- and T-type calcium channel blocker lomerizine alleviated some of the changes in areas vulnerable to secondary degeneration. Lomerizine reduced morphological disruption, oxidative stress and phosphacan expression, and limited early increases in macrophage numbers. However, lomerizine failed to prevent progressive de-myelination of ON axons. Within the retina, secondary retinal ganglion cell (RGC) death was significant in areas vulnerable to secondary degeneration. Lomerizine protected RGCs from secondary death at 4 weeks but did not fully restore behavioural function (optokinetic nystagmus). We conclude that blockade of calcium channels is neuroprotective and limits secondary degenerative changes following CNS injury. However such an approach may need to be combined with other treatments to ensure long-term maintenance of full visual function.
Asunto(s)
Traumatismos del Nervio Óptico/tratamiento farmacológico , Piperazinas/farmacología , Degeneración Walleriana/tratamiento farmacológico , Animales , Axones/efectos de los fármacos , Axones/metabolismo , Axones/patología , Bloqueadores de los Canales de Calcio/farmacología , Bloqueadores de los Canales de Calcio/uso terapéutico , Canales de Calcio Tipo L/efectos de los fármacos , Canales de Calcio Tipo L/metabolismo , Modelos Animales de Enfermedad , Femenino , Gliosis/tratamiento farmacológico , Gliosis/etiología , Gliosis/patología , Macrófagos/efectos de los fármacos , Macrófagos/patología , Fibras Nerviosas Mielínicas/efectos de los fármacos , Fibras Nerviosas Mielínicas/metabolismo , Fibras Nerviosas Mielínicas/patología , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Nervio Óptico/efectos de los fármacos , Nervio Óptico/patología , Nervio Óptico/fisiopatología , Traumatismos del Nervio Óptico/patología , Traumatismos del Nervio Óptico/fisiopatología , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Piperazinas/uso terapéutico , Ratas , Proteínas Tirosina Fosfatasas Clase 5 Similares a Receptores/metabolismo , Células Ganglionares de la Retina/efectos de los fármacos , Células Ganglionares de la Retina/metabolismo , Células Ganglionares de la Retina/patología , Resultado del Tratamiento , Tubulina (Proteína)/efectos de los fármacos , Tubulina (Proteína)/metabolismo , Degeneración Walleriana/patología , Degeneración Walleriana/fisiopatologíaRESUMEN
BACKGROUND/AIMS: While the glucocorticoid triamcinolone acetonide (9alpha-fluoro-16alpha-hydroxyprednisolone, TA) has been widely administered as a treatment of ocular inflammation, mineralocorticoids have not been tested for their efficacy. METHODS: We assessed cellular morphology and actin distribution by immunomicroscopy and light microscopy, membrane permeability with transendothelial resistance and cell surface vascular endothelial growth factor receptor-1 (VEGF-R1) expression by flow cytometry. RESULTS: Fludrocortisone acetate was more effective than TA in restoring quiescent morphology and reducing membrane permeability in phorbol-12-myristate-acetate (PMA)-stimulated choroidal endothelial cells (CECs). Each of the corticosteroids inhibited VEGF-R1 cell surface expression in PMA-responsive CECs. CONCLUSION: Mineralocorticoids may be of potential use in reducing vascular permeability in ocular disease.
Asunto(s)
Coroides/citología , Células Endoteliales/efectos de los fármacos , Mineralocorticoides/farmacología , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo , Actinas/metabolismo , Animales , Permeabilidad Capilar/efectos de los fármacos , Línea Celular Tumoral , Tamaño de la Célula/efectos de los fármacos , Cortodoxona/farmacología , Relación Dosis-Respuesta a Droga , Citometría de Flujo , Regulación de la Expresión Génica/efectos de los fármacos , Macaca mulatta , ARN Mensajero/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular/genética , Acetato de Tetradecanoilforbol/farmacología , Triamcinolona Acetonida/farmacologíaRESUMEN
Scarring after severe burn is a result of changes in collagen deposition and fibroblast activity that result in repaired but not regenerated tissue. Re-epithelialisation of wounds and dermal cell repopulation has been thought to be driven by cells in the periphery of the wound. However, recent research demonstrated that cells originating from the bone marrow contribute to healing wounds in other tissues and also after incisional injury. We investigated the contribution of bone marrow-derived cells to long-term cell populations in scar tissue (primarily fibroblasts and keratinocytes) after severe burn. Wild-type mice were lethally irradiated and then the bone marrow reconstituted by injection of chimeric bone marrow cells expressing EGFP marker protein. Mice with chimeric bone marrow were then given a burn, either an 1-cm diameter injury (to mimic minor injury) or 2-cm diameter (to mimic moderate injury). Wounds were analysed at days 1, 3, 7, 14, 21, 28, 56 and 120 using FACS and immunohistochemistry to identify the percentage and cell type within the wound originating from the bone marrow. The inflammatory cell infiltrate at the early time-points was bone marrow in origin. At later time-points, we noted that over half of the fibroblast population was bone marrow-derived; we also observed that a small percentage of keratinocytes appeared to be bone marrow in origin. These findings support the theory that the bone marrow plays an important role in providing cells not only for inflammation but also dermal and epidermal cells during burn wound healing. This increases our understanding of cell origins in the healing wound, and has the potential to impact on clinical practice providing a potential mechanism for intervention away from conventional topical treatments and directed instead to systemic treatments affecting the bone marrow response.