Studies On The Mechanisms Of Bone Marrow Stromal Cells Treatment Of Stroke

Bone marrow stromal cells (MSC) are isolated from adult bone marraw. They are multipotential, and can differentiate into various tissue lineages, including bone, chondracytes, fat, myocytes, neurons and hepacytes etc. MSC can tranform to myocyte, neuron and hepocyte, etc. MSC can benefit the restoration and regeneration of injued tissues. After transplantation of human MSC, very few MSC can directly transform to the tissue cells, and the injured tissues can not restore by these transfomation. MSC secrets and/or promote the injured tissue to secret many bio-active factors then changing the microenvironment and repair the injured tissues.It was found that MSC can improve neurological function by promoting synaptic plasticity, neurogenesis and angiogenesis during the treatment of stroke in the research according to Dr Chopp and his coworkers’s work in recent20years. The mechanisms of MSC treatment include reducing the expression of inhibitory proteins in astrocytes which will ihibit neurite outgrowth, extension and transference of MSCs, promoting brain, especial astrocytes to secret neutrophic factors, growth factors and trophic factors. They also found tissue plasminogen activator(tPA) plays a key role during MSCs treatment of stroke. After MCAo mice were injected with MSCs intravenously, MSC can significantly increase tPA expression and activity in injured brain tissue, and consequently contributes to neurological function improvement.Sonic hedgehog (Shh) pathway is highly conserved. Shh acts as morphogens during embryonic development and involves in the repair of brain tissue injure and neurogenesis. The in vitro data verified MSC can increase Shh expression in astrocytes, and Shh pathway activation can subsequently cause the increase of tPA and decrease of plasminogen activator inhibitor1(PAI-1). Until now there is no in vivo research to investigate how MSC increases tPA expression and activity in vivo. Therefore the mechanisms that MSCs causes tPA increase is still unclear.Based on the above results, the hypothesis was raised that the Sonic hedgehog (Shh) signal transduction pathway promotes the MSC mediated activation of tPA in parenchymal cells. This activation of tPA in parenchymal cells increases neurite remodeling. To test this hypothesis, first, this subject measured the temporal profiles of cellular tPA expression and Shh expression in astrocytes of WT-mice subjected to middle cerebral artery occlusion with MSC treatment, or Shh pathway blocker--cyclopamine treatmen, or combination of MSC and cyclopamine treatment. These mice were also evaluated of neurological funtion, neurogenesis, axon outgrowth and synaptic regeneration. Secondly, exogeous Shh was intraventricularly infused to MCAo mice, the effects of direct administration of Shh were observed including neurite outgrowth and the protein expression of tPA and PAI-1in ischemic brain. The aims were to find the pathway which increase tPA expression and activity, investigate the role of Shh, and find the origin of Shh which may exist between the interaction of MSC and brain parenchymal cells--it is from exogenous MSC or from brain parenchymal cell secretion and dissect the relationship between Shh and tPA, during MSC treatment of stroke.To examine whether in vivo blockage of the endogenous Shh pathway affects grafted MSC induced neurite remodeling and functional recovery, adult C57BL/6mice subjected to middle cerebral artery occlusion (MCAo) were administered cyclopamine, a specific inhibitor of Shh receptor, smoothened (Smo), with and without MSC treatment. Four groups of mice were, respectively, treated with:1) MSC(n=12);2) vehicle (Control, n=12);3) cyclopamine (CP), n=9; and4) MSC and cyclopamine (MSC-CP, n=12). Neurological functions were tested at1,3,7, and14days after MCAo. Electron microscopy and immunofluoresence staining were employed to measure synapse density, and protein expression of tissue plasminogen activator (tPA) and Shh in parenchymal cells, respectively. To further confirm that Shh increases tPA expression and neurite remodeling, recombinant mouse Shh was intraventricularly infused into mice subjected to MCAo starting from day1post stroke. Immunostaining of phosphorylated and non-phosphorylated neurofilament (NF) and Western blotting of tPA and plasminogen activator inhibitor1(PAI-1) protein expression in the ischemic boundary zone (IBZ) were performed. In vitro studies with primary neurons were performed and neurite branch number and length and the Shh concentration in the medium were measured to complement in vivo data.In vivo, MSC treatment promoted functional recovery and neurite outgrowth after focal cerebral ischemia, concurrent with increase of synaptophysin expression, synapse density and Bielschowsky silver and luxol fast blue expression along the IBZ. Double immunostaining showed a significant increase in tPA and Shh expression primarily in astrocytes in the MSC group compared to Control. After combination treatment with MSC-CP or CP mono-treatment, the above effects were reversed. Exogenous Shh treatment after MCAo significantly increased neurite remodeling and tPA level and decreased PAI-1level compared to Control. In vitro, MSC co-culture gain and loss of function of Shh experiments on neurite outgrowth further confirmed and complemented in vivo data.In conclusion, MSC treatment improves neurological functional recovery, neurite outgrowth, and simutaneous causes tPA expression increase in stroke. Shh mediates the neurological function recorery. MSC first increased Shh gene expression, and then caused Shh protein expression increase. Shh is the upstream signaling of tPA. MSC administration activated Shh pathway, and Shh subsequently increased the gene and protein expression of tPA. The increase of Shh came from brain parenchymal cells directly, not from MSC secretion in local brain in tissue.