| Backround and ObjectiveBrain stroke,including ischemia and hemorrhage,is one the most important reasons that cause death and disabilities.The effects of current therapies are very limited.In the last few decades,however,the idea of being able to repair the brain by introducing new cells has become an accepted potential treatment strategy.Various cellular sources such as ES-derived neurons,neural stem cells(NSCs) from fetal or adult brain, MSCs derived form various tissue have been studied for stroke models.Among these, MSCs may be the most prospective cell sources,because they have significant advantages such as the lack of ethical controversy regarding their derivation and potential for autologous transplants.At first,the goal over a decade ago was to promote cells grafting to repair lost neuronal connections and conductivity.However,although considerable studies have been done,there is little or no evidence or true differentiation and integration and functional replacement of neural cells bu stem cells in the injured brain.Rencently,an increasing number of studies suggest that exogenous cells serve as a source of trophic support,promoting endogenous repair such as neurogenesis, angiogenesis,and synaptogenesis.Still yet,a third goal is to prevent ongoing cell death, dampen inflammatory response,.reduce the scar formation and potentially promote the remodeling of the white matter of the brain.Several clinical safety trials of stem-cell-based therapies are ongoing in stroke,and most are about MSCs.Our team are also preparing for the clinical trial for stroke with MScs.However,we think that there are still some important issues to address before embarking on clinical trials:(1) the mechanisms underlying cell therapy-mediated function recovery remains not fully understood,the investigations should be going on;(2) studies about the time window,the transplant approach and the dose response should be done to detect the appropriate answer;(3) MSCs can be derived form the patient's own bone marrow for autologous transplant,but patients with stroke are usually old patients, the rate of deriving MSCs from bone marrow of elder ones tends to be low,and the expedition in vitro is also less effective,considering the low immunogencity,allogenic transplant might be easier to practise,and most of the animal experiments have to use MSCs form exogenic or allogenic too;But there are few experiments designed for transplant immunity about MSCs.In this present study,we designed a series of experiments to explore the lowest effective dose of MSCs in brain transplant therapy for hemorrhagic rodent stroke models, and the mechanisms underlying the MSCs-mediated function recovery.We also try to investigate the problem about exogenic transplant immunity of MSCs,and to find the possibility and effectivity of repeated MSCs brain transplants.MethodsThis study consisted of two parts:in prat I,with a collagenase-induced intracerebral hemorrhage rodent stroke model,we designed a series of experiments to explore the lowest effective dose of MSCs and the mechanisms underlying the MSCs-mediated function recovery.The animals were randomly divides into 4 group:A 2×10~5/15ul (1×10~6/kg),B 4×10~4/15ul(2×10~5/kg),C 8×10~3/15ul(4×10~4/kg),D:contrast.Behavior testing,histological examination including immunofluorescent staining,HE staining and Nissl staining,RP-PCR analysis were done for evaluation the function recovery and detected the therapy mechanisms.In part II,with the peripheral blood monocytes as contrast,we investigated the survivals of the human bone marrow-derived MSCs transplanted into the rat's brain for twice,and evaluated the inflammatory response of the injection location,and compared the functional recovery of models groups treated once and twice.We also did mixed lymphocytes co-culture to detect the immunogenicity of MSCs.Results1.Behavior testing indicated obviously improvement in group A,and minor improvement in group B only at the time points of 3 days and 1 week after MSCs transplant.No improvement was detected in group C.2.There was no difference between the groups about the volume of the hematoma;but the inflammatory response was evidently dampened by MSCs(decreasing of the ED+or MPO+cells around the hematoma);comparing with the contrast group,the RT-PCR analysis demonstrated lower expression of IL-iβ,IL-2 and TNF-a3.Apoptosis was obviously reduce in the MSCs transplant group;Nissl staining also showed more neurons.4.Cells transplanted tended to migrate towards the lesion and aggregate around the hematoma;MSCs tended to promote angiogenesis.5.Exogenic transplanted MSCs were not immune rejected,and did not initiate specific immune response during the second transplant.6.Better recovery was detected by twice transplant;we also found that MSCs transplanted 2 weeks later,was also helpful in the behavior recovery.7.MSCs could not stimulate the proliferation of exogenic lymphocytes,however,they suppressed the lymphocytes proliferation stimulated by ConA.ConclusionOur results showed MSCs infused into brain cortex can improve neurofunctional recovery in rat collagenase-induced intracerebral hemorrhage.And the lowest effective dose was between 2×10~5/15ul(l×10~6/kg) and 4×10~4/15ul(2×10~5/kg).The transplanted stem cells can survive and migrate towards the lesion.Dampening the inflammatory response,preventing apoptosis and promoting angiogenesis might be underlying mechanism.Exogenic transplanted MSCs were not immune rejected,and did not initiate specific immune response during the second transplant. |
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