Experimental Study Of Recombinant Human Granulocyte Colony-Stimulating Factor On Influence To Changes Of Surrounding Haematoma After Intracerebral Hemorrhage In Rats

Intracerebral hemorrhage (ICH) is a common disease in the nervous system characterized by high mortality,relapse rate and unability. Since the pathogenesis of ICH is not very clear and there are no fundamental therapeutic methods, it has become one of hot points on research. Animal experiments and clinical researches have confirmed that mechanism of apoptosi plays a important role in brain tissue damage after ICH. Mechanism of apoptosi is closely related to course and prognosis of ICH Around haematoma. It has great sinificance for promoting the recovery of nerve function and guiding clinical teatment that a further research on signaling mechaism of apoptosi and how to reduce apoptosi in early stage after ICH.Discovering of stem cells(SCs) and continual research to its function are at the frotier of medical field and even life science. Stem cell therapy is the most perspective to solving regeneration of neurons currently and is called the Final Therapy of Brain Injury. Cell transplanting becomes the best therapeutic regimen to dysfunction caused central nervous system because it is the most promising to recovering the normal functions of nervous system. Bone marrow stem cells (BMSCs) mobilized by recombinant human granulocyte colony-stimulating factor (rhG-CSF) has the partial advantages of SCs and can overcome hard problem of SCs transplant technology. It is one of the reasonable method in treatment of cerebrovascular disease.Objective: In the present, by establishing the stable and reliable animal model of ICH in Wistar rats, we observed the pathological changes of tissues around lesion after rhG-CSF treatment comparing to contral group. After that, we approached the dynamic changes of Caspase-3 and STAT3 in brain from protein level and genetic level by the advanced techniques and researched interrelation between dysfunction of nerve system and their expression.We approached the dynamic changes of Brdu in brain by immunohistochemistry method in order to observe mobilization capability of rhG-CSF to BMSCs. We ivestigated the effect and mechanism on apoptosi in ICH rats after rhG-CSF treatment.It can provide theoretical basis and new clinical therapeutic direction on ICH by cell transplanting.Methods: Wistar rats were used to create ICH models by injecting blood to caudate putamen under sterological method.1d, 3d, 7d and 14d after operation we performed neurological deficit score on rats and observed the pathological changes of brain tissue around lesion stained by HE method under light microscope. Expression of Caspase-3, STAT3 and Brdu were investigated by immunohistochemistry at each time point and RT-PCR was used to approach the relationship between Caspase-3 and STAT3 expression .Results: Neurological deficit score: Contral group and treatment group appearanced dysfunction of nerve system and sham operation group did not appearanc. Score of contral group and treatment group had no significant change at 1d after operation, but score of treatment group was rather lower than contral group at 3d, 7d and 14d after operation(P<0.05).The pathological changes under light microscope: In contral group, At 1d after haematoma, brain tissues around lesion became loose and matrix had the slight edema, which became obvious at 3d with the broken and necrosis of Nissl's body, and karyopyknosis. Neutrophil, swollen astrocytes, myelin and axon disaggregation could be found, a various sizes of vacuoles between cells, neutrophil and lymphocyte cell infiltration were obvious, which reached the peak at 3d. From 7d to 14d, the edema deceased gradually and proliferated glia repaired, vacuoles and neutrophil between cells were seldom at 7d. Matrix had little edema, proliferated glia repaired and neuronal degeneration were visible at 14d. In treatment group, the structure was still complete at 1d after injection. matrix had the little edema. Lymphocyte cells and moncyte cells occurred around haematoma, necrosis and inflammatory in treatment group were alleviated than In contral group,peaked at 3d. Proliferated glia repaired and neuronal degeneration were fewer number than contral group at 7d, and edema lessened . At 14d, the organization structure tended to tidniness, the edema disspeared, and necrosis of neurons were not obvious.The results of immunohistochemistry: In contral group, we observed a few sporadic Caspase-3 positive neurons and gliocytes at 1d. Sham operation group occurred few positive cells. We found caspase-3 positive cells in the endotheliocytes seldomly. The Caspase-3 positive cells peaked at 3d, concentrated on circumvascular cells. then decreased gradually. At 14d a few positive cells could still be found. Caspase-3 expression level was obvious higher in contral group than that in treatment group at 3d, 7d and 14d, there was significant difference of the number of positive cells in treatment group compared to contral group at 3d, 7d and 14d (P<0.01). The same way In contral group, we observed a few sporadic STAT3 and Brdu positive neurons and gliocytes at 1d. Sham operation group occurred few positive cells. We found that STAT3 and Brdu positive cells arranged in disorder, increased at 3d, STAT3 and Brdu positive cells peaked at 7d, then decreased gradually at 14d . STAT3 and Brdu positive cells expression level were obvious lower at each time point in contral group than that in treatment group, there was significant difference of the number of positive cells in treatment group compared to contral group (P<0.01). We found STAT3 and Brdu positive cells in the endotheliocytes seldomly in contral group,but concentrated on circumvascular cells. then decreased gradually. At 14d a few positive cells could still be found. STAT3 and Brdu expression level was obvious higher in treatment group than that in contral group at at each time point, there was significant difference of the number of positive cells in treatment group compared to contral group at each time point (P<0.01). During the whole period, there were a lot of STAT3 and Brdu positive endotheliocytes in treatment group . STAT3 and Brdu increased range obviously in treatment group compared to contral group at each time point.RT-PCR results indicated that the products electrophoresis all were positive expressed at 485bp or 375bp after the target gene of Caspase-3 and STAT3 by RT-PCR amplification. Caspase-3mRNA and STAT3mRNA all were highly expressed in treatment group and in contral group. The number of Caspase-3 expression increased at 1d, peaked at 3d, then decreased gradually. Nevertheless, the number of STAT3 heightened at 1d, peaked at 7d, then decreased gradually. We can still observe lower Caspase-3 expression level and higher STAT3 expression level in treatment group than in contral group at each time point. The results from RT-PCR were consistent to the results from immunohistochemistry.Conclusions: To injected autologous blood into caudate putamen is an ideal model for ICH and double injection method has its advantages because of high rate of haematoma formation, good repeatability and strong stability. This model for ICH is worth popularizing. Apoptosis mechanism plays a very important role in brain injury after intracerebral hemorrhage. The treatment of recombinant human granulocyte colony-stimulating factor can decrease Caspase-3 expression level and promote STAT3 expression level ,consequently reduces apoptosis of neurons. Recombinant human granulocyte colony-stimulating factor can mobilize efficiently bone marrow stem cells to alleviate neuronal differentiation and enhance the repairment of neuronal cells after intracerebral hemorrhage, so it si considered to have neuroprotecttive effect.This experimental study provided a therapeutic foundation for clinical treatment ICH by using cell transplantation in future.