Granulocyte-colony stimulating factor protects against endoplasmic reticulum stress in an experimental model of stroke.

Granulocyte-colony stimulating factor (G-CSF) is an endogenous growth factor that exhibits a diverse range of neuroprotective mechanisms against a variety of neurological disorders including ischemic stroke. We investigated the anti-apoptotic mechanisms of G-CSF against endoplasmic reticulum (ER) stress induced apoptosis. Sprague-Dawley rats were subjected to transient occlusion of the middle cerebral artery (MCAO) for 90 min. Rats were injected with G-CSF (n = 15; 50 µg/kg body weight s.c.) for 4 days, starting 24 h post-MCAO and brains were harvested after 4 days reperfusion (n = 16). Key proteins in ER stress apoptosis were analyzed by immunoblotting. G-CSF reduced infarct volume to 53% and improved neurological deficits. G-CSF treatment significantly (P < .05) attenuated the expression of proteins involved in ER stress apoptosis pathway; ATF4, ATF6, p-p38MAPK, pJNK and CHOP. G-CSF treatment also re-established ER homeostasis evident by the reduction of the intraluminal ER stress sensor, GRP78 as well as reducing the overall cellular stress level protein, HSP27. G-CSF also up-regulated anti-apoptotic proteins pAKT and Bcl-2 while down-regulated the pro-apoptotic protein Bax. G-CSF exerts neuroprotection from cerebral ischemia through the preservation of the ER, resulting in the attenuation of pro-apoptotic proteins and the potentiation of anti-apoptotic proteins.

Mode of action of S-methyl-N, N-diethylthiocarbamate sulfoxide (DETC-MeSO) as a novel therapy for stroke in a rat model.

One approach for protecting neurons from excitotoxic damage in stroke is to attenuate receptor activity with specific antagonists. S-Methyl-N, N-diethylthiocarbamate sulfoxide (DETC-MeSO), the active metabolite of disulfiram, has been shown to be a partial antagonist of glutamate receptors and effective in reducing seizure. First, we investigated neuroprotective effect of DETC-MeSO on primary cortical neuronal culture under hypoxia/reoxygenation condition in vitro. Then, DETC-MeSO was administered subcutaneously for 4 and 8 days with the first injection occurring 1 h before or 24 h after reperfusion in the rat middle cerebral artery occlusion stroke model. Rats were subjected to the neuroscore test, and the brain was analyzed for infarct size. Monitoring neurotransmitter release was carried out by microdialysis. Heat shock proteins, key proteins involved in apoptosis and endoplasmic reticulum (ER) stress, were analyzed by immunoblotting. DETC-MeSO greatly reduced both cell death following hypoxia/reoxygenation and brain infarct size. It improved performance on the neuroscore test and attenuated proteolysis of αII-spectrin. The level of pro-apoptotic proteins declined, and anti-apoptotic and HSP27 protein expressions were markedly increased. Levels of the ER stress protein markers p-PERK, p-eIF2α, ATF4, JNK, XBP-1, GADD34, and CHOP significantly declined after DETC-MeSO administration. Microdialysis data showed that DETC-MeSO increased high potassium-induced striatal dopamine release indicating that more neurons were protected and survived under ischemic insult in the presence of DETC-MeSO. We also showed that DETC-MeSO can prevent gliosis. DETC-MeSO elicits neuroprotection through the preservation of ER resulting in reduction of apoptosis by increase of anti-apoptotic proteins and decrease of pro-apoptotic proteins.

Improvement of contusive spinal cord injury in rats by co-transplantation of gamma-aminobutyric acid-ergic cells and bone marrow stromal cells.

BACKGROUND AIMS: Cell therapy is considered a promising option for treatment of spinal cord injury (SCI). The purpose of this study is to use combined therapy of bone marrow stromal cells (BMSCs) and BMSC-derived gamma-aminobutyric acid (GABA)ergic inhibitory neurotransmitter cells (BDGCs) for the contusion model of SCI in rats. METHODS: BDGCs were prepared from BMSCs by pre-inducing them with ß-mercaptoethanol followed by retinoic acid and then inducing them by creatine. They were immunostained with BMSC, proneuronal, neural and GABA markers. The BDGCs were intraspinally transplanted into the contused rats, whereas the BMSCs were delivered intravenously. The animals were sacrificed after 12 weeks. RESULTS: The Basso, Beattie and Bresnahan test showed improvement in the animals with the combined therapy compared with the untreated animals, the animals treated with GABAergic cells only and the animals that received BMSCs. The immunohistochemistry analysis of the tissue sections prepared from the animals receiving the combined therapy showed that the transplanted cells were engrafted and integrated into the injured spinal cord; in addition, a significant reduction was seen in the cavitation. CONCLUSIONS: The study shows that the combination of GABAergic cells with BMSCs can improve SCI.

Induction of bone marrow stromal cells into GABAergic neuronal phenotype using creatine as inducer.

PURPOSE: Deficits involving GABAergic neurons have been reported in aging, central nervous trauma and neurodegenerative disorders; bone marrow stromal cells (BMSCs) have been proposed as a feasible source of donor cells in replacement cell therapy. In this study, the effects of creatine on transdifferentiating BMSCs into GABAergic-like neurons were evaluated in vitro. METHODS: The BMSCs were isolated from adult rats, preinduced by ß-mercaptoethanol (BME) and retinoic acid (RA), and then induced by creatine into GABAergic-like neurons. The cells were characterized using different differentiation markers. The functionality of the differentiated cells was evaluated using FM1-43. RESULTS: The isolated cells expressed Oct-4 and were immunoreactive to fibronectin and CD44. The highest percentages of cells immunoreactive to nestin and neurofilament-68 were found at the second day of preinduction. At the induction stage, there were increases in the number of cells immunoreactive to neurofilament-200, MAP-2, synapsin I, synaptophysin, and NeuN. The percentages of the immunoreactive cells to GABAergic neuron markers increased. The optimal induction dose was 5 mM. The dose of 10 mM showed a decline in the expression of most of these markers. The functionality test indicated that the synaptic vesicles were released upon stimulation. CONCLUSION: Creatine can induce the differentiation of BMSCs to the GABAergic neuronal phenotype within one week.

In vitro transdifferentiation of bone marrow stromal cells into GABAergic-like neurons.

BACKGROUND: Cell therapy of many neurodegenerative diseases using bone marrow stromal cells (BMSC) requires the differentiation of BMSC into neuronal subtype. However, the transdifferentiation of BMSC into GABAergic phenotype requires more investigation. METHODS: In this study, BMSC of adult female rats were pre-induced into neuroblast-like cells using 1 mM beta-mercaptoethanol (betaME) and 10 microM retinoic acid (RA), followed by 40 mM potassium chloride as inducer. The BMSC were evaluated by fibronectin as well as Oct-4. The percentage of nestin, neurofilaments (NF 68, NF 160, and NF 200) and GABA immuno-reactive cells was used to evaluate the GABAergic differentiation at the pre-induction and induction stages. The statistical analysis was carried out using unpaired student's t-test and ANOVA with Tukey's multiple comparison. RESULTS: The BMSC in the fourth passage expressed fibronectin up to 91.24 +/- 0.82%. The pre-induced cells after 2 days of RA exposure showed the expression of neuroblastic markers of nestin and NF68 (81.56 +/- 2.64% and 82.12 +/- 2.65%, respectively). The yield of GABAergic neurons with beta-ME for 1 h and RA as pre-inducer for 2 days followed by potassium chloride as inducer (40 mM for 3 days) was 60.64% +/- 1.97%. In addition, NF160 and NF200 were detected in the transdifferentiated cells. RT-PCR showed no expression of Oct-4 after the induction and pre-induction stages. CONCLUSION: GABAergic-like neurons obtained from BMSC can be potentially used in cell transplanting for some neurodegenerative disorders.