| Background Mesenchymal stem cells (MSCs) are stem cells that lying in marrow .The research showed that the cells could survive and were induced to divide so as to be confirmed as neural stem cells (NSCs) in vivo and ex vivo as so. It could be used to replace the neural cells or repair the nervous system, such as ischemic stroke, brain injury and so on. MSCs was easily grained and cultured, and there were various method of such as vein transplantation, directed transplantation. It provides a hopeful method for central nervous system diseases. The objective of this study is to probe the separation in vitro culture and expanding method of mesenchymal stem cells (MSCs), and investigate the biological characteristics of MSCs by observing the morphous and growth velocity of MSCs, and identification of the phenotype of MSCs. Method MSCs were isolated and purified from human being using density centrifugation and anchoring culture, then cultured in low-glucose DMEM supplement with 10% fetal bovine serum (FBS) for amplification. MSCs were identified by observing the morphous and growth velocity of MSCs, identification of the phenotype of MSCs. Draw the growth curve of MSCs and determine the mitotic index of MSCs. Observe the survival rate and growth state of MSCs underwent freeze thawing.Result MSCs with stable biological properties can be efficiently isolated and purified by density centrifugation and adhering to the culture plastic flask and expanded satisfactory in L-DMEM medium with 15% fatal calf serum. MSCs got together at 9th-12th day .The passage MSCs proliferated fast. MSCs were purified at 3rd generation. MSCs grew in whirlpool. Through detection by flow cytometry, the expressions of CD29 were positive; the exprssions of CD34 and CD45 were negative. As the passage increases, the cloning efficiency and proliferative ability of MSCs decrease. The survival rates of MSCs thawed after being frozen 1 month and 6 months were 90% and 85% respectively.Conclusion hBMSCs with stable biological properties can be efficiently isolated and purified by density centrifugation and adhering to the culture plastic flask and expanded satisfactory in L-DMEM medium with 15% fatal calf serum. Freeze thawing has no obvious influence on MSCs. BackgroundSpontaneous ICH causes 10 to 20% of all strokes, but effective and standardized clinical treatment remains elusive. By the very nature of its pathophysiological features, spontaneous ICH results in a variety of neural injury mechanisms: direct mechanical injury, ischemia, toxicity, and apoptosis. No currently available medical therapy has shown a consistent or unambiguous benefit in terms of functional outcome. In recent years attention has been focused on the ability of undifferentiated pluripotent stem cells to improve experimental neurological conditions, including ischemic stroke, brain trauma, and spinal cord injury. Specifically, human embryonic neural stem cells have been used in a collagenase model of ICH to restore neurological function and demonstrate migration of the cells to the site of hemorrhage.Bone marrow contains a subpopulation of cells that can serve as tissue stem cells because they can be used as precursors of nonhematopoietic tissue. These pluripotent cells of bone marrow origin are referred to as MSCs. The MSCs have a capacity for self-renewal and differentiation in a variety of nonhematological tissues, and have the potential to be used for cell therapy. In the appropriate cellular microenvironments, MSCs are able to produce mesenchymal tissues, such as fibrous tissue, bone, cartilage, and muscle, and can differentiate specifically into adipocytes, osteoblasts, and chondrocytes. Of significance for the treatment of neurological disorders, MSCs pass through the blood-brain barrier to target sites of brain lesions under experimental conditions. In the neonatal mouse, MSCs migrate widely throughout the developing brain and have shown the capacity to differentiate into neurons and astrocytes. Cells of bone marrow origin infused systemically into rats preferentially migrate to ischemic cortex. In recent studies, hBMSCs have shown significant benefit in animal models of ischemic stroke and closed head injury. In these models of neural damage, the MSCs appear to have the capacity to induce endogenous brain-derived cells, likely derived from the SVZ, to participate in the restorative process. In light of the recognized ability of intravascularly delivered MSCs to treat neural injury and the potential application of stem cell technology to treat ICH, this experiment was conducted to test the hypothesis that hBMSCs improve functional outcome and reduce cellular injury after experimental ICH. Methods Animal PreparationEighty adult male Wistar rats weighing between 270 and 320g were used for this study. Stereotactic stabilization and localization were used after general anesthesia was induced with 3 ml/kg chloral hydrate. A 1-mm craniectomy was performed and the stereotactically guided needle was placed at coordinates 0.5 mm anterior, 3.5mm lateral, and 5.5 mm deep relative to the bregma. The ICH was induced by injecting 2 ul saline solution (consisting of IV type collagenase 0.4U and heparin 4U) into the right striatum, with a steady infusion rate of 0.5 ul/minute. Experimental GroupsThe rats were randomly divided into four experimental groups. Group 1, which consisted of 20 animals, was given 1 × 10~6 hBMSCs in 1 ml PBS carrier solution, slowly injected into the rats' tail veins; Group 2, which consisted of 20 animals, was given 3 ×10~6 hBMSCs in PBS solution; Group 3, which consisted of 20 animals, was given 6×10~6 hBMSCs in PBS solution; and Group 4, control group, which was given the 1 ml PBS vehicle solution as a placebo. For mitotic labeling of newly formed DNA, all rats also received daily intraperitoneal injections of 100 mg/kg BrdU starting 24 hours after ICH and continuing for the next 13 days. Assessment of Neurological FunctionNeurological function was evaluated using the NSS, which were performed before and at 1, 7, and 14 days after ICH. The NSS is a composite score in which motor, sensory, balance, and reflex measures are used to calculate a value ranging from 1 to 18, with the higher score implying greater neurological injury. All animals were killed after 14 days, and their brains were fixed in formalin and sliced into 2-mm-thick sections. Every 40th coronal section (cut at a thickness of 6 am between the bregma +0.1 mm to -0.86 mm in each rat, for a total of six sections) was used for H & E and immunochemical staining.The percentage of striatal tissue loss in one section was calculated using an image analysis system.The area of preserved striatum on the side of the hemorrhage was subtracted from the area of the contralateral striatum, thus reckoning the degree of encephalomalacia or tissue loss from the injury in that brain section. A percentage value was then calculated by dividing the amount of cell loss by the total area of the contralateral striatum.Well-established immunohistochemical analytical methods were used, which consisted of staining both control and treatment groups with synaptophysin, TUJ1, mAb 1281, and BrdU. Synaptophysin is a marker of presynaptic plasticity and synaptogenesis; TUJ1 is a developmental neuronal marker, whereas mAb 1281 is specific for all human cell types and is used to identify hBMSCs. On the other hand, BrdU (100 mg/kg) is a marker for newly formed DNA and is generally accepted as an expression of cell division and new cell growth. Control experiments consisted of staining coronal brain tissue sections as outlined earlier, but omitted the use of primary antibodies. Quantification of TUJ1, BrdU, mAb 1281and SynaptophysinFor semiquantitative measurements of TUJ1, and Synaptophysin, six slides from the block (bregma +0.1 mm to — 0.86 mm) were used. Synaptophysin was measured in the striatum, and TUJ1 were measured at the SVZ. Synaptophysin,and TUJ1 were digitized under a 20 xobjective lens by using a 3-CCD color video camera interfaced with an MCID image analysis system. Data are presented as a percentage of area, in which the TUJ1- and synaptophysin-immunopositive areas in each field were divided by the total areas in the field (628 ×480 um~2). The BrdU-positive cell number was measured in the boundary around the lesion. Quantitative data for mAb 1281 are presented as the total number of mAb 1281-immunoreactive cells within contralateral and ipsilateral areas of each slide. Statistical AnalysisStatistical evaluations of functional scores, area of ICH-related tissue damage, and histochemical results were performed using the independent Student t-test. ResultsAll 80 animals survived the 14-day experimental period. There was no apparent difference between the control group and any of the experimental groups in the results of NSS and corner turn tests 1 day after ICH. Nevertheless, after 7 days both tests showed significant improvement in results for the rats injected with hBMSCs compared with controls.The area of tissue loss as a percentage of the normal hemisphere was as follows (given as the mean±standard error of the mean for all values): control, 30±1.1%; 1 million hBMSCs,23±2.7% (p = 0.002); 3 million hBMSCs, 23 ±2.5% (p = 0.003); and 6 million hBMSCs, 23± 3.9% (p = 0.002).As can be seen, there is virtually no difference between any of the treatment groups, with all of them showing significant improvement over the control group 2 weeks after ICH.The mAb 1281, BrdU, Synaptophysin and TUJ1 histochemical staining data suggest that there was a significant increase in the positive-staining cells in the region of ICH for all treatment groups compared with the controls. Specifically, for TUJ1, and synaptophysin labeling, the area of positive-staining cells was significantly increased in treated animals compared with controls. Labeling of mAb 1281 was seen in the treated animals in the region of the ICH, verifying that the injected hBMSCs did reach the site of injured brain preferentially compared with the contralateral hemisphere. Staining for BrdU was significantly increased in the boundary zone around the ICH, implying localized new cell formation in the rats treated with hBMSCs compared with control animals. Control immunostaining, which omitted the primary antibodies, did not show positive-staining cells. ConclusionsIntravenous injection of hBMSCs at doses of 1, 3 and 6 million cells 1 day after experimental ICH improves neurological function and is associated with a significant reduction in local tissue loss.By 14 days posttreatment, the injected human cells are found in high concentrations at the site of the hemorrhage. There were significant increases in immature neurons, neuronal migration, synaptogenesis, and new cell formation in the striatum and SVZ near the site of the ICH in the animals receiving the hBMSCs.This improvement in the treated animals is associated with reduced tissue loss and increased local presence of the hBMSCs, mitotic activity, immature neurons, synaptogenesis, and neuronal migration. BackgroundSpontaneous IVH is an infrequent but severe complication of hemorrhage stroke. Morality rate has been reported to be as high as 42.6 to 83.3%, morality rate of surgery 35.7 to 100%. When IVH is large enough to impede normal CSF circulation, acute obstructive hydrocephalus can occur in the subacute and chronic stages of IVH, communicating hydrocephalus may develop if fibrosis of the basal leptomeninges occurs or reabsorption of CSF becomes impaired from fibrosis of the arachnoid villus. Although there have been some medical or surgical therapies for IVH, none of them are encouraging. Among the frequently used are: ①external ventricular drainage (EVD) combined with urokinase; ② hematoma evacuation of craniotomy with bone flap or bony opening;③evacuation and drainage of intraventricular hematoma with stereotactic operation. EVD combined with urokinase offers a simple operation technique with less injury on cerebral cortex, and no further injury on the deep nucleus mass. Patients thus have a quick recovery after operation. It is especially fit for patients older in age or with multiple organ dysfunctions. It also can be applied to treatment for intraventricular hemorrhage in pregnancy. However, secondary infection and rebleeding etc may happen due to less accuracy in setting catheters, longer drainage time, catheter easier to get blocked and poorer effect of pressure reduction. Craniotomy with bone flap can evacuate hematoma deep in the brain and intraventricular hematoma, with a complete reduction of pressure. On the other hand, however, this operation technique may cause too much traction to cortex, and is likely to aggravate injury to deep nucleus mass. Craniotomy with small bony opening simplifies the operation, and can evacuate and remove hematoma with pincers under direct vision. No active hemorrhage may occur if there is no forced evacuation of blood-clots on the perisporium and bottom of hematoma cavity. However, it does not help minimize operative injury since it is difficult to accurately locate the hematoma. Evacuation and drainage of intraventricular hematoma with stereotactic operation place great emphasis on protection for the cortex and deep nucleus mass. Pressure reduction does not have a good effect sometimes since the operation is not conducted under direct vision and rebleeding rate might be 4 to 10% with more difficulty in evacuating hard blood clots; Injury of negative pressure to brain tissue cannot be avoided completely when aspiration is conducted. The purpose of this study was to probe a method of minimally invasive treatment for intraventricular hemorrhage, i.e. a neuroendoscopic approach to IVH. Methods1 Patients Forty-two patients with IVH were treated surgically in our department between December 2002 and February 2004. A CT scan was performed on all patients preoperatively. ICH hematoma volume was measured on the head CT scan with the use of the ABC/2 method, in which A is the greatest diameter on the largest hemorrhage slice, B is the diameter perpendicular to A, and C is the approximate number of axial slices with hemorrhage multiplied by the slice thickness. Angiography was obtained when intracerebral aneurysms or arteriovenous malformations (AVM) were suspected.All patients were screened and enrolled if an IVH with or without ICH had occurred within 48 hours before admission and was diagnosed by clinical and brain CT criteria, and ICH volume is <30ml. In addition, when intracerebral aneurysms or arteriovenous malformations were suspected, they had to be excluded by appropriate diagnostic studies. The patients presenting with IVH resulting from cerebellar and brainstem hemorrhage were excluded from this study. This study is prospective and randomized. All patients were divided into external ventricular drainage (EVD) group and neuroendoscope (NE) group randomly, underwent external ventricular drainage and neuroendoscopic operation respectively.2 Operation In NE group, the neuroendoscopic operation was performed with patients in supine or latericumbent position within 48 hours from onset under general anesthesia.For patients presenting with spontaneous primary or secondary IVH (ICH volume <20ml), operative access was precoronal or postcoronal by a 30 mm burr hole. The incision was 3-4 cm long. The dura was opened in a cross. A variable-angled (0° or30°) rigid neuroendoscope with an outer diameter of 6 mm was introduced into monolateral or bilateral ventricle.If there is less intraventricular hematoma, with bigger clearance leftover inside ventricle, and with hematoma partially liquefied, the neuroendoscope was used alone and the procedure performed through the endoscopic channels (EN, endoscopic neurosurgery). Aspiration was alternated with irrigation with normal saline and was promptly stopped when the whitish color of the ventricular walls appeared.If there is large amount of intraventricular hematoma, with smaller clearance leftover inside ventricle and with hematoma adhered to the wall of ventricle, microneurosurgical techniques and the endoscope were used in combination, with part of the operation being carried out with the assistance of the endoscope (NEAMN, neuroendoscopic assisted microneurosurgery). Suction tube is inserted close by the endoscope to evacuate hematoma by means of better lighting ,clear and enlarged visual field provided by the endoscope. In case of bleeding, bipole coagulator can be used for hemostasis.Once the chorodial plexus and Monro foramen were identified, the instrument was advanced into the third ventricle to remove hematoma.Finally, the endoscope was flexed toward the frontal or occipital horn and the trigonus to evacuate these sections from the clots. Leave the endoscope inside ventricle for 5-10minutes and withdraw the endoscope after making sure there is no bleeding. At the end of the procedure, monolateral or bilateral ventriculostomy was performed in all patients by placing an intraventricular catheter (IVC) for both ICP monitoring and drainage (with a constant gradient of 15 mm Hg).Within 24 hours and 1 week after surgery, a CT scan was obtained respectively.For patients without any significant remaining intraventricular hematoma, the external ventricular diversion was kept open for 2 to 3 days. On pressure stabilization, it was removed. If findings show that the remaining IVH volume is>10ml, the patient should receive injection of urokinase (UK) into the ventricle. The UK preparation made in Tianjin Biochemical pharmaceutical factory was a sterile, lyophilized preparation intended for intraventricular injection .A vial of UK contains 250,000IUas powder, which is reconstituted with sterile normal saline to yield a solution that contains 25,000IU of UK per milliliter. Patients received a dose that ranged from 25,000IU to 30,000IU of UK. After injection, the IVC was closed for 1 hour to prevent drainage of UK away from the clot and to allow adequate time for drug-clot interaction. After 1 hour of closure, the IVC was reopened with an appropriate drainage gradient. The UK was administered every 8 hours, for about one week until the IVC was removed based on the patient tolerance to IVC closure for 24 hours (i.e., no sustained ICP elevation > 15 mm Hg).For patients presenting with spontaneous secondary intraventricular hemorrhage (ICH volume >20ml), the operative approach was selected based on the projection of intracerebral hematoma on skull surface by means of CT scan image. Remove intracerebral hematoma and by its breaking into ventricular route remove intraventricular hematoma according to the method described above. Postoperative treatment was the same as that described above.In EVD group, traditional external ventricle drainage with dissolving hematoma with urokinase was performed with patients in supine position within 48 hours from onset under local anesthesia. Conventional puncture into lateral ventricle was adopted from forehead. Postoperative treatment was the same as that described above. 3 Therapeutic Evaluation, Follow-Up and Statistical analysisAfter 2 months of treatment, grading was concluded based on Glasgow outcome scale (GOS) put forward by Jennett and Bond in 1975.Excellent (5 points): fully independent with no residual disability. Good (4 points): independent with moderate disability. Fare (3 points): significant disabilities requiring assistance in most daily life activities. Poor (2 points): persistent vegetative state. Dead (1 points): death.Clinical follow-up was at 2 months (GOS).Chi-square test and Fisher's exact test of probabilities were applied to evaluate the GOS score. ResultsIn Neuroendoscope group, CT scan 24 hours after surgery showed that, almost compete removal (> 90%) of intracerebral or /and intraventricular hematoma was achieved in 15 cases, < 90% removal in 7 cases. There was not intracranial infection and rebleeding after surgery in all cases.In EVD group, CT scan 24 hours after surgery showed that, partial removal (< 60%) of intraventricular hematoma was achieved in 3 cases, almost no removal in 17 cases. There was no rebleeding in all cases. Intracranial infection after surgery was observed in 2 cases.All patients were followed up for two months and evaluated at two months from surgery according to GOS. The follow-up results were listed in Table 1.The result was dead in 2 cases of neuroendoscope group (died of pneumonia and acute myocardial infarction respectively), and in 2 cases of EVD group (died of pneumonia and pneumonia with stress ulcer respectively). Patients in EVD group, compared with neuroendoscope group, showed poor recovery after two months of surgery. The excellence and goodness rate (ratio between number of excellent and good patients and number of all patients in this group) in EVD group is < in Neuroendoscope group, and the difference between two groups is statistically significant (x~2 =4.752, P<0.05).The difference in mortality rate between two groups was not statistically significant (x~2 =0.010, P>0.05).Conclusions Neuroendoscopic neurosurgery for intraventricular hemorrhage offers better surgical treatment because it is characterized by visualized manipulation, effective hemorrhage evacuation and excellent postoperative outcomes.
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