Mesenchymal Stem Cell Grafts Engineered To Release Adenosine Ameliorate Seizures And Cognitive Deficts In Epileptic Rats

Objective:To observe lentiviral RNAi mediated downregulation of adenosine kinase (ADK) in rat mesenchymal stem cells (rMSCs), and represent cells for subsequent experiment.Methods:After lentiviral transduction of rMSCs, we determine the optimal multiplicity of infection based on GFP expression and cell morphology. Following lentiviral transduction of rMSCs with anti-ADK RNAi, immunofluorescence and western bloting were applied to analysis downregulation of ADK in mesenchymal stem cells.Results:Rat bone marrow mesenchymal stem cells obtained in whole bone marrow adherent culture method were uniform morphological, with less fusiform cells and high activity, the optimal MOI of lentiviral transduction of MSCs was 30 and transducted efficiency was 93.6±4.2%. Following lentiviral transduction of MSCs with anti-ADK RNAi, we demonstrate up to 53% downregulation of ADK and a concentration of 13.8 ng adenosine per ml of medium after incubating 105 cells for 8 hours.Conclusions:RNA interference (p236) can significantly downregulate ADK in mesenchymal stem cells, thereby increasing the content of adenosineObjective:In this part,we aim to investigate the antiepileptic and neuroprotective effect of mesenchymal stem cell grafts engineered to release adenosine in lithium- pilocarpine model of epilepsy.Methods:MSCs with a knockdown of ADK or cells expressing a control sequence were transplanted into bilateral hippocampus of rats 1 week prior to the intraperitoneal injection of pilocarpine. A control group of ADK' -MSCs recipients received DPCPX (lmg/kg, i.p.)and maintained throughout the course of this study.in acute phase (1 day) and chronic phase (30 days),we test behavioral detection (SRS frequency monitoring), electrophysiology (EEG) and immunohistochemistry(Nissl staining, TUNEL staining) to study the changing of brain electrical activity and neuronal damage.Results:While rats with control implants expressing a control sequence(SHAM) were characterized by pilocarpine induced status epilepticus and subsequent hippocampal neuronal cell loss, animals with therapeutic ADK knockdown implants reduced the level V seizure duration in half an hour of status epilepticus and a reduction in spontaneous recurrent seizure; a reduction in the number of TUNEL-positive cells and the activity of caspase-3 one day after SE, and 33% reduction in hippocampus neuronal cell loss 30 days after SE, This effect was reversed by receiving DPCPX.Conclusions:RNAi mediated knockdown of ADK in hippocampal MSC implants is an effective strategy to reduce seizures and neuronal loss in a rat model of pilocarpine, and seizure suppression is due to graft mediated adenosine release acting via A1Rs.Objective:In this part,we aim to investigate seizure-associated cognitive deficits effect of mesenchymal stem cell grafts engineered to release adenosine in lithium- pilocarpine model of temporal lobe epilepsy.Methods:MSCs with a knockdown of ADK or cells expressing a control sequence were transplanted into bilateral hippocampus of rats 1 week prior to the intraperitoneal injection of pilocarpine. A control group of ADK- -MSCs recipients received DPCPX (lmg/kg, i.p.)and maintained throughout the course of this study.in 2 week and 4 week, we test behavioral detection (Morris Water maze and Novel Object Recognition Test) to study seizure-associated cognitive deficits effect of mesenchymal stem cell grafts engineered to release adenosine in lithium-pilocarpine model of temporal lobe epilepsy.Results:In Morris Water maze test, control TLE rats showed significant deficits in learning and memory in comparison to naive rats. This deficit was reduced after ADK'-MSCs transplantation, while this reducing was abolished receiving DPCPX. In novel object recognition test, an independent measure of learning and memory, control TLE rats showed significantly decreased time exploring the novel object in comparison to the naive rats, this deficit was reduced after ADK"-MSCs transplantation, whereas this effect was reversed after receiving DPCPX. The frequency of novel object exploration showed a similar trend as the duration of novel object exploration but did not reach statistical significance.Conclusions:Lithium- pilocarpine- induced temporal lobe epilepsy rats present cognitive damage; mesenchymal stem cell grafts engineered to release adenosine ameliorate cognitive deficits in epileptic rats and due to grafts mediated adenosine release acting via A1Rs.