tretinoin and Brain-Injuries

Peretinoin is an acyclic retinoid that stimulates retinoic acid receptors (NR1Bs) and produces therapeutic effects on hepatocellular cancer. We have previously shown that NR1B agonists such as Am80 and all trans-retinoic acid suppress pathogenic events in intracerebral hemorrhage. The present study addressed the actions of peretinoin and Am80 against cytotoxicity of a blood protease thrombin on cortico-striatal slice cultures obtained from neonatal rat brains. Application of 100 U/ml thrombin to the slice cultures for 72 h caused cell death in the cortical region and tissue shrinkage in the striatal region. Peretinoin (50 μM) and Am80 (1 μM) counteracted these cytotoxic effects of thrombin, and the effect of peretinoin and Am80 was blocked by LE540, an NR1B antagonist. A broad-spectrum kinase inhibitor K252a (3 μM) attenuated the cytoprotective effect of peretinoin in the cortical region, whereas a specific protein kinase A inhibitor KT5720 (1 μM) attenuated the protective effect of peretinoin in the cortical and the striatal regions. On the other hand, nuclear factor-κB (NF-κB) inhibitors such as pyrrolidine dithiocarbamate (50 μM) and Bay11-7082 (10 μM) prevented thrombin-induced shrinkage of the striatal region. Peretinoin and Am80 as well as Bay11-7082 blocked thrombin-induced nuclear translocation of NF-κB in striatal microglia and loss of striatal neurons. We also found that daily administration of peretinoin reduced histopathological injury and alleviated motor deficits in a mouse model of intracerebral hemorrhage. These results indicate that NR1B agonists including peretinoin may serve as a therapeutic option for hemorrhagic brain injury.

Topics: Animals; Antineoplastic Agents; Brain; Brain Injuries; Cerebral Hemorrhage; Mice; NF-kappa B; Rats; Thrombin; Tretinoin

The effect of substances known as inducers of neuronal differentiation on cultured human and mouse adipose-derived mesenchymal stem cells (ASCs) and their fate after transplantation into the injured and ischemic mouse brains were studied. ASCs were isolated from the human and mouse adipose tissue. Inducers of neuronal differentiation included β-mercaptoethanol, glial cell line-derived neurotrophic factor (GNDF), brain-derived neurotrophic factor (BDNF), retinoic acid (RA), 5-azacytidine, as well as their combinations. Three days after the induction, the phenotype of the induced cells was analyzed using immunocytochemistry and real-time PCR assay for differential expression of specific genes. The induction efficiency was evaluated by the increased transcription of neuronal differentiation markers: nestin, β-III-tubulin (Tub-B), microtubule-associated protein 2 (MAP2), and neuron-specific enolase (ENO2). The expression of marker genes was tested by immunocytochemical analysis. ASC cultivation in the medium with RA or BDNF in combination with 5- azacytidine for a week increased the mRNA and protein levels of nestin, Tub-B, and ENO2. The transplantation of induced mouse ASCs into the mouse brain increased the lifespan of the cells relative to control uninduced cells and promoted their migration from the transplantation site to the recipient cerebral parenchyma. The transplantation of the induced cells into the mouse brain pre-exposed to endothelin- 1 promoted a more active cell migration into the surrounding ischemic brain tissue. Thus, ASC exposure to RA or BDNF in combination with 5-azacytidine elevated the transcription of the neuronal differentiation markers and improved the viability and integration of ASCs grafted into the mouse brain.

Topics: Adipose Tissue; Adult; Animals; Azacitidine; Brain Injuries; Brain-Derived Neurotrophic Factor; Cell Differentiation; Doublecortin Domain Proteins; Glial Cell Line-Derived Neurotrophic Factor; Humans; Intermediate Filament Proteins; Male; Mercaptoethanol; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Mice; Microtubule-Associated Proteins; Middle Aged; Nerve Tissue Proteins; Nestin; Neurons; Neuropeptides; Phosphopyruvate Hydratase; Receptor, trkB; Tretinoin; Tubulin

We have treated undifferentiated mouse embryonic stem (ES) cells with all-trans retinoic acid (RA) to induce differentiation in vitro into neuron-like cells with good cell viability for use as a graft. Furthermore, we asked whether the RA-induced neuron-like cells restored neurological dysfunction. To this end, the cells were transplanted into right hemiplegia model of mice, developed by a cryogenic injury of motor cortex. Motor function of the recipients was gradually improved, whereas little improvement was observed in control mice. The lesion showed clustering of mature and almost mature neuron-like cells in mice transplanted with the RA-treated cells. The grafted cells had synaptic vesicles. This finding may suggest their maturation and synaptic connection in the recipient brain. Even though further study is necessary to elucidate molecular and cellular mechanisms responsible for the functional recovery, we consider that the ES cells may have advantage for use as a donor source in various neurological disorders including motor dysfunction.

Topics: Animals; Antineoplastic Agents; Brain Injuries; Brain Tissue Transplantation; Cell Differentiation; Endothelium, Vascular; Hemiplegia; Mice; Mice, Inbred C57BL; Motor Cortex; Movement; Neurons; Recovery of Function; Stem Cell Transplantation; Stem Cells; Tretinoin

Human Ntera-2 (NT2) cells can be differentiated in vitro into well-characterized populations of NT2N neurons that engraft and mature when transplanted into the adult CNS of rodents and humans. They have shown promise as treatments for neurologic disease, trauma, and ischemic stroke. Although these features suggest that NT2N neurons would be an excellent platform for ex vivo gene therapy in the CNS, stable gene expression has been surprisingly difficult to achieve in these cells. In this report we demonstrate stable, efficient, and nontoxic gene transfer into undifferentiated NT2 cells using a pseudotyped lentiviral vector encoding the human elongation factor 1-alpha promoter and the reporter gene eGFP. Expression of eGFP was maintained when the NT2 cells were differentiated into NT2N neurons after treatment with retinoic acid. When transplanted into the striatum of adult nude mice, transduced NT2N neurons survived, engrafted, and continued to express the reporter gene for long-term time points in vivo. Furthermore, transplantation of NT2N neurons genetically modified to express nerve growth factor significantly attenuated cognitive dysfunction following traumatic brain injury in mice. These results demonstrate that defined populations of genetically modified human NT2N neurons are a practical and effective platform for stable ex vivo gene delivery into the CNS.

Topics: Animals; Brain Injuries; Cell Differentiation; Female; Gene Expression Regulation; Gene Transfer Techniques; Genetic Therapy; Genetic Vectors; Graft Survival; Green Fluorescent Proteins; Humans; Lentivirus; Luminescent Proteins; Mice; Mice, Nude; Nerve Growth Factor; Neurons; PC12 Cells; Peptide Elongation Factor 1; Rats; Recovery of Function; Stem Cell Transplantation; Stem Cells; Treatment Outcome; Tretinoin

Degenerative diseases represent a severe problem because of the very limited repair capability of the nervous system. To test the potential of using stem cells in the adult central nervous system as "brain-marrow" for repair purposes, several issues need to be clarified. We are exploring the possibility of influencing, in vivo, proliferation, migration, and phenotype lineage of stem cells in the brain of adult animals with selective neural lesions by exogenous administration (alone or in combination) of hormones, cytokines, and neurotrophins. Lesion of the cholinergic system in the basal forebrain was induced in rats by the immunotoxin 192IgG-saporin. Alzet osmotic minipumps for chronic release (over a period of 14 days) of mitogens [epidermal growth factor (EGF) or basic fibroblast growth factor (bFGF)] were implanted in animals with behavioral and biochemical cholinergic defect and connected to an intracerebroventricular catheter. After 14 days of delivery, these pumps were replaced by others delivering nerve growth factor (NGF) for an additional 14 days. At the same time, retinoic acid was added to the rats' food pellets for one month. Whereas the lesion decreased proliferative activity, EGF and bFGF both increased the number of proliferating cells in the subventricular zone in lesioned and nonlesioned animals. These results are indicated by the widespread distribution of BrdUrd-positive nuclei in the forebrain, including in the cholinergic area. Performance in the water maze test was improved in these animals and choline acetyltransferase activity in the hippocampus was increased. These results suggest that pharmacological control of endogenous neural stem cells can provide an additional opportunity for brain repair. These studies also offer useful information for improving integration of transplanted cells into the mature brain.

Topics: Acetylcholine; Animals; Brain Injuries; Bromodeoxyuridine; Cell Differentiation; Cell Division; Cell Movement; Choline O-Acetyltransferase; Epidermal Growth Factor; Fibroblast Growth Factor 2; Male; Maze Learning; Mitogens; Nerve Growth Factor; Neurons; Rats; Rats, Sprague-Dawley; Stem Cells; Tretinoin