Bone Marrow-derived Mesenchymal Stem Cells Treated Intracerebral Hemorrhage And Toxicity Of Single Administration In The Brain Of Macaca Fascicularis

Background and ObjectiveIntracerebral hemorrhage(ICH) is one of the common diseases which hurt the people.In China,ICH accounts about 25～30%of all cerebral stroke patients. Although the rate is not very high,the mortality of ICH is 2～6 times that of the ICD. Many survivals have remaining neurological impairments.ICH can induce direct destroy to the brain tissue.The brain tissue and nerve fibers are compressed and displaced,deformed or discontinued.The active materials delivered from the hemorrhage produce cytotoxicity,breakdown the blood brain barrier,induce brain edema.Ischemia and hypoxia induce the death of nerve cells and discontinue of the nerve fiber.Angiogenesis and neuroprotection are preconditions to implement the nerve remodeling.Flk1~+ human bone marrow-derived mesenchymal stem cells have the ability to differentiate into endothelial cells.Vascular endothelial growth factor (VEGF) can stimulate neovascularization.And other neurotrophic factors such as brain-derived neurotrophic factor(BDNF) and nreurotrophic-3(NT-3) can also have the ability to protect nerves.Bone marrow-derived mesenchymal stem cells(BMSC),sourcing from mesoderm,are multipotent and represent one group ofnon-hematopoietic stem cells in the marrow,which can differentiate into multiple lineages,such as nerve cells,bone, cartilage or fat.Flk1~+ hBMSC have the ability to differentiate into endothelial cells. Because BMSC can be easily obtained,quickly amplificated ex vivo,autoplastic transplanted,induce immune tolerance,relieve or inhibit graft-versus-host diseases. So BMSC was widely used in the preclinical or clinical research in nerve system disease,autoimmune diseases or blood diseases.How long can the stem cells survive and what is the exact mechanism of the treatment? Is there a certain information connection between transplanted hBMSC and host neurons? There are no consensuses about all these questions.There are still a lot of controversial issues about BMSC therapy for stroke in the preclinical research.There are many difficulties to solve on the effect and safety of the cell therapy.We confirmed the efficacy of hBMSC treated rat ischemia in our prophase work.But great disparation exists between species.We cannot conclude that hBMSC are also effective and safe in the treatment of nonhuman primates.So,in present study we designed a series of experiments to explore the effects and the mechanism of BMSC transplantation to macaca fascicularis with ICH in order to supply the preclinical data for the stem cell therapy.MethodsThe study includes four parts:1.To set up macaca fascicularis ICH animal model by injection of autoallergic femoral artery blood.And the model was evaluated by stroke neurological deficits score for monkeys,MRI,PET and pathology.2.The monkeys were divided into two groups,acute-phase-therapy group and chronic-phase-group.Using stroke neurological deficits score,MRI,PET, immunofluorescence technique,pathology to estimate the availability and possible mechanism of stem cell therapy.3.hBMSC labeled with superparamagnetic iron oxide(SPIO) were injected into the cortex of normal and ICH monkeys.Tracing the cells in vivo by Magnetic resonance imaging(MRI) aim to study the survival and migration of BMSC.4.We got the therapeutic dose by the three parts of work above and continue to do the toxicity experiment of hBMSC.By the test of general state of monkeys,stroke neurological deficits score,the routine and biochemistry exam of cerebrospinal fluid (CSF) and blood,MRI and pathological exam of organs,we confirmed the safety of hBMSC single administration.Results1.The macaca fascicularis ICH model was successly set up by three-injection method.Using neurological deficit score for monkeys,PET,MRI,pathology,we found the model can well analogue the pathological change of ICH.2.After hBMSC transplantation,PET reveals standardized uptake value(SUV) heightened in the areas of cortex and basal ganglion nucleus surrounding ICH.It is obvious in 2～3 weeks after BMSC transplantation.Magnetic resonance spectroscopy (MRS),MRI and pathology display that after hBMSC transplantation,nerve tissue necrosis was lessened,and hecatomb was absorbed more quickly than the control. Immunofluorescence,microvessel density measurement,PET and pathology show that many new vessels formation beside ICH after hBMSC transplantation.3.MRI can display hBMSC 12 weeks after transplantation in the brain of macaca fascicularis.But we did not found obvious migration of stem cells.4.Single injection of hBMSC into the brain of macaca fascicularis had no toxicity to nerve function,CSF,blood,or organs.ConclusionThe macaca fascicularis model was successly set up by three-injection method and it can analogue the pathological change of ICH.hBMSC transplantation can promote the formation of new vessels,increase the blood flow of brain tissue,protect nerve tissue,and enhance the absorption of hematoma and functional recovery of monkeys.SPIO can effectively label hBMSC and MRI can trace transplanted stem cells in vivo.Single administration of hBMSC into the brain of macaca fascicularis had no toxicity.